Composite flash-precipitated nanoparticles

ABSTRACT

The invention described herein relates to sterically stabilized colloidal constructs comprising preformed colloidal particles encapsulated within a polymeric shell. The constructs, which are controllably sized, are nanoparticles comprising hydrophobic elements, electrostatically charged particles with hydrophobic surfaces, hydrophobic inorganic nanostructures, and amphiphilic copolymers with hydrophobic domains and hydrophilic domains. The constructs are made by a process that allows for the simultaneous encapsulation of a preformed colloidal agent as well as a dissolved hydrophobic active within the core of the polymeric nanoparticle. Among the actives incorporated in various embodiments are organic fluorescent dyes, metal nanostructures and superparamagnetic materials for use in combined fluorescence, optical and magnetic resonance imaging applications, and hydrophobic drugs for therapeutic applications.

GOVERNMENT RIGHTS IN THIS INVENTION

This invention was made with government support under Grant No.CMS0609049 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

FIELD

The invention relates to the field of nanotechnology. In particular, theinvention is embodied in a hydrodynamic process for manufacturing ahydrophilic carrier, preferably less than 1000 nanometers in size, thatencapsulates hydrophobic agents or electrostatically charged agents withhydrophobic surfaces, including pharmaceuticals, organic imaging agentsor fluorescent dyes, and solid inorganic nanostructures.

BACKGROUND

Nanoparticles have become increasingly important in the development ofnew materials for enhanced drug delivery and imaging applications(Adams, M. L. et al., J. Pharmaceutical Sciences 2003, 92:1343-1355;Portney et al., Analytical and Bioanalytical Chemistry 2006,384:620-630). Drug carriers such as liposomal (Kim, S. Drugs 1993,46:618-638), polymeric vesicle (Discher et al., Science 2002, 297,967-973) and micellar dispersions (Allen et al., Colloids and SurfacesB—Biointerfaces 1999, 16:3-27; Kwon, Critical Reviews in TherapeuticDrug Carrier Systems 1998, 15:481-512) consisting of particles 50-400 nmin diameter have shown great promise, for example, in the formulation ofanticancer therapeutics that would be highly insoluble in aqueous mediaabsent their incorporation into a carrier. Such carriers, besidesaffording more potent drug delivery, also provide opportunities forselective tumor targeting. More recently, inorganic nanoparticles,including quantum dots (Michalet et al, Science 2005, 307:538-544) goldnanospheres (West et al., Annual Review of Biomedical Engineering 2003,5:285-292), nanoshells (Loo et al. Cancer Research and Treatment 2004,3:33-40), and superparamagnetic metals (Mornet et al., Journal ofMaterials Chemistry 2004, 14:2161-2175) have been explored fornanoparticle-based biomedical functions, such as tagging, medicalimaging, sensing, and separation.

Despite extensive innovation over the last decade, there remains a needfor integrated, easily adaptable drug delivery and imaging modalities,especially those for the delivery and monitoring of highly toxiccompounds in vivo. Polymeric nanoparticles in particular are a versatilemedium for this purpose, due to their enhanced drug loading capacity,biological stability, and extended in vivo circulation times (Kwon etal., Advanced Drug Delivery Reviews 1995, 16:295-309).

Polymeric nanoparticles that carry drugs and other agents encapsulatedin their cores have evolved. Initially, research efforts focused oncombining polymeric carriers of drugs with organic fluorescent dyes forparticle visualization, without regard to the “encapsulation” of eitherthe drug or the dye. Fluorescent nanoparticless have been prepared bybinding water-soluble fluorophores to the surfaces of pre-formednanoparticles (O'Reilly et al., Journal of Polymer Science PartA—Polymer Chemistry 2006, 44: 5203-5217) or, more commonly, bychemically tethering a fluorescent dye to the hydrophobic terminus of anamphiphilic block copolymer and then permitting the polymer toself-assemble into a particle (Luo, et al., Bioconjugate Chemistry 2002,13:1259-1265). Organic dyes and fluorophores, however, require directvisualization, and so are generally practical only for in vitroapplications such as nanoparticle cellular uptake and localizationstudies (Savic et al., Science 2003, 300:615-618).

Nanoparticles having a metallic core that adds contrast to imagesacquired by magnetic resonance imaging, for example, or computed X-raytomography are more suitable for in vivo biomedical applications (Bulteet al., NMR in Biomedicine 2004, 2004, 17: 484-499; Hainfeld et al.,British Journal of Radiology 2006, 79:248-253). Typically, however, theyare incompatible with body fluids because their surfaces are hydrophobicand they may also be incompatible because of toxicity. A number ofcoating strategies have been used to address these issues (Azzam et al.,Langmuir 2007, 23:2126-2132; Kim et al., Langmuir 2007, 23: 2198-2202;Butterworth et al., Colloids and Surfaces A—Physicochemical andEngineering Aspects 2001, 179: 93-102; Gupta et al., Biomaterials 2005,26: 3995-4021; Soo et al., Langmuir 2007, 23:4830-4836). Researchershave also functionalized the surfaces of such inorganic nanoparticleswith receptor-specific peptides or protein ligands, allowing fortargeted localization of the imaging particles (Paciotti et al., DrugDevelopment Research 2006, 67:47-54; Zhang et al., Biomaterials 2002,23:1553-1561; Zhou, et al., Biomaterials 2006, 27:2001-2008). Also,ligands (optionally together with drugs) can be covalently attached tothe coating material instead of to the inorganic nanoparticle itself(Paciotti et al., Drug Delivery 2004, 11:169-183; Yu et al., Journal ofMaterials Chemistry 2004, 14: 2781-2786; Gupta et al., Biomaterials2004, 25:3029-3040). Since the coating material is advantageouslyhydrophilic, however, the strategy of attaching hydrophobic moieties(e.g., drugs) to it is generally not practical.

SUMMARY

In one embodiment, the present invention contemplates a process formanufacturing composite nanoparticles, the process comprising:

-   -   a. providing an organic compound dissolved in a solvent,    -   b. providing an inorganic nanoparticle dispersed in the same        solvent,    -   c. providing an amphiphilic polymer dissolved in the same        solvent, and    -   d. mixing the solvent mixture with an anti-solvent such that a        composite nanoparticle forms, the composite nanoparticle        comprising the organic compound, the inorganic nanoparticle and        the amphiphilic polymer.

In preferred embodiments, the organic compound is hydrophobic andinsoluble in water, the inorganic nanoparticle is hydrophobic, and thesolvent is a water-miscible organic solvent such as tetrahydrofuran,dimethyl sulfoxide, or ethanol.

In some embodiments, a first solvent in which the nanoparticle isdispersed, a second solvent in which the organic compound is dissolvedand a third solvent in which the polymer is dissolved are provided andmixed with the anti-solvent under conditions such that the compositenanopraticle forms.

The inorganic nanoparticle may be surface-modified prior to step dabove. It may also be functionalized to give the nanoparticle ahydrophilic surface. The organic compound may be electrostaticallycharged prior to step d, as may the inorganic nanoparticle.

In preferred embodiments, the invention provides a dispersion ofcomposite nanoparticles that does not flocculate in an aqueous solvent.

The amphiphilic polymer may be selected from the group consisting of anycopolymer, block copolymer, graft copolymer, comb-graft copolymer, andrandom copolymer that contains both hydrophobic and hydrophilic regionswithin the same copolymer.

The inorganic nanoparticle may be selected from the group consisting ofmagnetic, paramagnetic and superparamagnetic metals, and oxides thereof,or from the group consisting of gold, palladium and oxides thereof, orit may be a quantum dot.

In an embodiment especially preferred for use in magnetic resonanceimaging, the inorganic nanoparticle comprises an iron oxide. In a mostpreferred embodiment, the iron oxide comprises CoFe₂O₄. In someembodiments, the iron oxide is a nanocrystal.

Preferably, the organic compound and the inorganic nanoparticle areencapsulated in a hydrophobic core region of the composite nanoparticle,and a hydrophilic shell surrounds the core. Encapsulated compounds andparticles may be stabilized therein by means of steric hindrance,electrostatic charge stabilization or a combination thereof.

Some embodiments of the invention comprise a composite nanoparticle,said nanoparticle comprising an organic compound, preferablyhydrophobic, an inorganic nanoparticle, which may be metallic ornon-metallic, and an amphiphilic copolymer. The composite nanoparticlemay comprise a pharmaceutical composition, said pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluent.

In some embodiments, the inorganic nanoparticle is an imaging contrastagent which may be selected, without limitation, from the group ofmetals consisting of Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II),Fe(III), Pr(III), Nd(III), Sm(III), Tb(III), Yb(III), Ho(III), Eu(II),Eu(III), Er(III), Indium (In), Technetium (Tc), and Barium or from thegroup of non-metals consisting of Iodine (I), Bromine, Fluorescein,Carboxyfluorescein and Calcein.

The composite nanoparticle may further comprise a targeting agent, whichmay be anchored on the external surface of the hydrophilic shell. Insome embodiments, the encapsulated organic compound or the inorganicnanoparticle are releasable from the composite nanoparticle. In someembodiments, the released compound, the released nanoparticle, or both,have targeting properties.

In some embodiments, the composite nanoparticle may further comprise atherapeutic agent embodied in the organic compound, the inorganicnanoparticle, the targeting agent or the amphiphilic copolymer of thecomposite nanoparticle.

In some embodiments, the invention provides a method of in vivo imagingof a site of disease in a subject, including without limitation a tumor,atherosclerotic plaque, an anatomic anomaly, or a benign lesion, themethod comprising administering to the subject the compositenanoparticle.

In some embodiments, the invention provides a method of determining thedistribution of the therapeutic agent in a subject being treated withthe therapeutic agent as a function of the distribution of an imagegenerated by the inorganic particle. In some embodiments, the image isan MRI image. In other embodiments, the image may be an X-ray image, ascintillographic image or an optical image.

In some embodiments, the composite nanoparticle, which is preferablyless than about 500 nm in mean intensity-average diameter, has adispersity index of less than about 0.3, and preferably less than about0.25. In some embodiments, the inorganic nanoparticles comprise at least1% of the weight of the composite nanoparticle, and may comprise up to50%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a process for preparing multicomponent(“composite”) nanoparticles by flash nanoprecipitation in a multi-inletvortex mixer, and a product thereof.

FIG. 2 shows the relationship between the size (hydrodynamic diameter)of PEG-b-PCL-encapsulated (“protected”) gold (“Au”) nanoparticles andthe amount of gold loaded therein. Figure inset demonstrates that sizedepends on the cubic root of Au volume fraction (φ_(Au)).

FIG. 3 compares size distributions of PEG-b-PCL protected Aunanoparticless at various Au loadings (in weight percent in finalsolution), and relative to polystyrene latex spheres.

FIG. 4 is a transmission electron micrograph of PEG-b-PCL (5,000-b-6,000g/mol)-protected Au nanoparticles prepared at a loading of 23.3 wt % Au.

FIG. 5A is a photograph of aqueous dispersions of PEG-b-PCL(5,000-b-6,000 g/mol) nanoparticles encapsulating Au (9.4 wt %) andβ-carotene (30.2 wt %), prepared via flash nanoprecipitation (vials 1and 2) compared with an ordinary (unprotected) gold colloid (vial 3).Vial (1) contains an unfiltered dispersion, vials (2) and (3) containfiltered dispersions.

FIG. 5B compares the light absorption spectra of the dispersions invials (1) and (2).

FIG. 6 compares particle size distributions of freshly made PEG-b-PCL(5,000-b-6,000 g/mol) nanoparticles encapsulating Au (9.4 wt %) andβ-carotene (30.2 wt %) in 155 mM NaCl (solid line) and nanoparticlesstored 28 days (dashed line) at room temperature.

FIG. 7 shows that increasing amounts of the encapsulating polymer in aPEG-b-PCL-protected Au nanoparticle do not add to the size of theparticle until the polymer component accounts for more than about 33% ofthe particle's volume.

FIG. 8 shows, for various concentrations of gold in solution, the sizedistribution of gold aggregates in the flash nanoprecipitation product(as formed in about 40 milliseconds), where the “size” of an aggregateis determined by the number of gold monomers in the aggregate.

FIG. 9 shows UV-visible absorbance spectra of dodecane capped Au(C₁₂—Au) nanoparticles dispersed in THF (solid line) and PEG-b-PCLprotected C₁₂—Au nanoparticles dispersed in 1:10 v/v THF:water (dashedline).

FIG. 10 is a graphical representation of the solubility of β-caroteneand a block copolymer stabilizer as a function of THF concentration at35° C.

FIG. 11 is scanning electron photomicrograph of representativenanoparticulate products.

FIG. 12 shows intensity-average particle size distributions, determinedby DLS, of PEG5-b-PCL9 protected CoFe₂O₄ CNPs prepared via FlashNanoPrecipitation.

FIG. 13 show DLS spectra of PEG5-b-PCL9 protected CoFe₂O₄ CNPs (3 wt %Fe) pre- and post-centrifugation and re-suspension.

FIG. 14 shows relaxation rates of magnetic moments of protons in watermeasured at 11.75 T and 25° C. in the presence of PEG5-b-PCL9 protectedCoFe₂O₄ CNPs.

FIG. 15 shows relaxation rates of magnetic moments of protons in watermeasured at 11.75 T and 298K in the presence of PEG-b-PCL stabilizedCoFe2O4 CNPs of identical composition.

FIG. 16 compares the intensity-average size distributions of PEG-b-PCLprotected CoFe₂O₄ particles and dually-loaded PEG-b-PCLCoFe₂O₄/β-carotene particles.

FIG. 17 shows relaxation rates of magnetic moments of protons in watermeasured at 11.75 T and 298K in the presence of PEG-b-PCL stabilizedCoFe₂O4 CNPs.

FIG. 18 shows T₂-weighted images of PEG5-b-PCL9 protected CoFe₂O₄ CNPsprepared at a Fe loading of 3.0 wt %.

DETAILED DESCRIPTION

One way of delivering hydrophobic materials in vivo would be toencapsulate hydrophobic moieties within a polymeric carrier that isexternally hydrophilic. This has proven to be a difficult problem whenone seeks to capture inorganic nanostructured solids in such a carrier,especially if the nanostructures are to be co-encapsulated withhydrophobic organic compounds.

Although the Applicants will not be bound by any theory of howembodiments of the instant invention work, it is widely understood that,in contrast to molecules and the atoms of ionic crystals such as sodiumchloride that dissolve in dilute solution and move about independentlyin (dilute) solution, nanostructures do not dissolve in a solvent asindividual atoms or molecules. The atoms of a nanostructure areorganized as a solid at the core. Solvents do not disrupt the core.Thus, nanoparticles disperse in solvents as particles, not as individualatoms or molecules. In part because of this, they attract one anotherreadily, and aggregate readily.

To make a nanoparticulate carrier that is a composite of hydrophobicorganic compounds and inorganic nanostructures, which carrier is toremain dispersed in an aqueous solvent, one is faced with finding a wayof (1) stabilizing dispersions of nanostructures in a local environmentthat is loaded with hydrophobic organic and inorganic agents (Liu etal., International Journal of Cancer 2007, 120: 2527-2537; Fahmy et al.,The AAPS Journal 2007, 9:E171-E180) and (2) stabilizing a population ofthe carriers as a dispersion in an aqueous solvent, bearing in mind thatthe carriers are themselves nanoparticles susceptible to aggregation.

The polymeric component of such a carrier suggests the possibility ofstabilizing dispersions within the carrier sterically; that is, byutilizing the polymer to build barriers between nanoparticles in thedispersions. The advantages of such delivery systems, viz., highdrug-loading capacity, reduced toxicity, protection of carried agentsfrom the surrounding environment, targeting, localization andmonitoring, and the possibility of tailoring the system's drug releasekinetics (Kwon et al., 1998; Soppimath et al., Journal of ControlledRelease 2001 70:1-20) are appreciated, but only a limited literature onthe successful preparation of hybrid organic-inorganic nanoparticleformulations exists. For example, Gao and coworkers (Nasongkla et al.,Nano Letters 2006 6:2427-2430) have described the preparation ofcomposite poly(ethylene glycol)-block-poly(D,L-lactide) micellesencapsulating chemotherapeutic doxorubicin and superparamagnetic ironoxide nanoparticles. More recently, the successful preparation ofantibody-conjugated poly(D,L-lactide-co-glycolide) nanoparticlesincorporating doxorubicin and magnetic iron nanocrystals was reported byYang et al. (Journal of Materials Chemistry 2007, Advance Article). Inboth cases, enhanced cancer cell affinity and improved magneticresonance signals were reported in vitro. Unfortunately, the preparativetechniques that were employed, namely solvent evaporation andemulsification processes, suffer from several disadvantages. First, theyrequire the use of stabilizing surfactants and numerous purificationstages to achieve only a low yield of uniformly sized nanoparticles.Additionally, hydrophobic components are relatively insoluble within theparticles, so they cannot host hydrophobic components at high capacity(Shuai et al., Journal of Controlled Release 2004, 98:415-426). Lastly,these preparative processes do not allow the artisan to independentlyspecify the amounts or kinds of individual components that the finalnanoparticle will carry. Constraining the artisan are the unequalsolubilities and miscibilities among the hydrophobic components, anynanostructures to be incorporated, and the hydrophobic domains of thestabilizing polymers. Furthermore, the processes do not ensure uniformdistribution of actives within the nanoparticulate carriers.

In its various embodiments, the present invention provides compositenanoparticles that carry organic and inorganic materials innanoparticulate “capsules” whose outer surfaces are compatible withaqueous solvents and remain homogeneously disperse in them.

In describing the embodiments, the following meanings attach to theterms employed. Unless otherwise noted, all terms of art, notations,scientific terms or other terminology have the meaning commonlyunderstood by persons of ordinary skill in the art to which theembodiments of the invention pertain, but may be defined herein forclarity or convenience.

As used herein, “a” or “an” means “at least one” or “one or more.”

The term “active agent” or “agent” refers herein to any chemical moietyor substance that has a desired behavior or activity. Non-limitingexamples include elements, inorganic or organic ions, molecules,complexes, particles, crystals and radionuclides that may be “active”(without limitation) as pharmaceuticals, as contrast agents in imagingapplications, as colorants, flavors, and fragrances, as sources orabsorbers of energy, as linking or binding agents, or as toxins.

As used herein, a “nanoparticle” is any object of a size less than about1 micron. It is not necessary that such a particle conform to this limitin all of its dimensions. Indeed, it is not even necessary that such aparticle have dimensions in the conventional sense. Quantum dots, forexample, may be referred to as “nanoparticles” herein. An “inorganicnanoparticle” as the term is used herein, generally refers to a particlecomprising a metallic element, and an “organic nanoparticle” generallyrefers to a particle comprising a polymer (nanoparticles structured fromelemental carbon are generally not regarded as “organic”). Particlescomprising a polymer and at least one other material may be defined as“composite nanoparticles,” but the term as used herein typically refersto nanoparticles constructed of a polymer, an inorganic nanoparticle,and another component that is neither a polymer nor an inorganicnanoparticle.

As used herein, “dissolved” or “molecularly dissolved” molecules oratoms are homogenously distributed in a solvent and move about thereinrandomly and largely independently of one another. A substance that is“insoluble in an aqueous medium” dissolves in solutions that arephysiologically relevant with respect to ionic strength, osmolality andpH only to the extent of 0.05 mg/ml or less. A solution that is“insoluble in pure water” dissolves in pure water only to the extent of0.05 mg/ml or less. The criterion for “soluble” on the other hand is 1.0mg/ml or more.

A “colloid” as used herein is analogous to a solution: both are systemsof molecules, atoms or particles in a solvent. The particles of acolloidal system, however, because of their size (nanometers) or thedistance between them (also nanometers), attract one another withsufficient force to make them tend to aggregate even when the only meansof transport for the particles is diffusion (the “diffusion-limitedregime”). As used herein, the term “colloidal particles” refers toparticles capable of forming a colloid. Although a “colloidal particle”is not itself a colloid but only a constituent of a colloid, the term“colloid” is often used to denote particles itself Thus, when thecontext so admits, the term “colloid” may refer herein to a particle.The term “colloidal dispersion” herein distinguishes colloids from truesolutions on the one hand and from “suspensions” of larger particles onthe other hand. In the latter, the particles tend to “settle out” likesand stirred in water. A colloid, in contrast, tends to “flocculate”when large aggregates of particles form in the dispersion. The terms“colloidal dispersion” and “colloidal suspension” are often usedinterchangeably and may be so used herein. Instead of being “dissolved”in a solvent, the particles in a colloidal dispersion are said to be“solubilized” in the solvent. The solvent may be referred to as a“continuous phase” and, more colloquially, as the “surroundingenvironment.”

As used herein, the term “emulsion” is a dispersion of liquid dropletsor liquid crystals in a liquid, wherein the droplets or crystals aregenerally larger than the particles in a colloidal system.

As used herein, a “polydisperse” colloid comprises particles that rangein size. In a “narrowly polydisperse” colloid, the range is small.

As used herein, “organic compounds” encompass the entire domain oforganic chemistry. Unless the context admits otherwise, however, organiccompounds are generally distinguished herein from polymers.

As used herein, a “hydrophobic moiety” is insoluble in aqueous solutionsas defined above. The “moiety” may be, without limitation, a smallmolecule, a nanoparticle, a polymer or a region of a polymer. Colloidalparticles may be hydrophobic or hydrophilic. Colloids (i.e., colloidaldispersions) may also be referred to herein as “hydrophobic” or“hydrophilic.” A colloidal dispersion comprising an aqueous continuousphase with hydrophobic particles dispersed therein is referred to as ahydrophobic colloid or hydrophobic dispersion. Hydrophobic dispersionsare thermodynamically unstable if the dispersion medium (or continuousphase) is aqueous. Conversely, a hydrophilic dispersion may be unstableif the dispersion medium is a non-polar solvent. Amphiphilic stabilizersmay be incorporated into such dispersions to counter the instability. Asused herein, an “amphiphilic stabilizer” is a compound having amolecular weight greater than about 500 grams/mole that has ahydrophilic region or domain and a hydrophobic region or domain.Preferably, the molecular weight is greater than about 1,000, or 1,500or 2,000, and may be much higher, e.g., 25,000 or 50,000 grams/mole.Preferably, an amphiphilic stabilizer is a polymer, and more preferablya polymer or polymer system that provides both hydrophobic andhydrophilic domains to the colloid Block copolymers, graft copolymers,comb-graft copolymers, and random copolymers that contain bothhydrophobic and hydrophilic regions within the same copolymer areuseful.

As used herein, the term “mixing” may refer, when the context so admits,to “micromixing,” which has a particular meaning herein as set forth indetail below.

Conventionally, the term “anti-solvent” relates to a solvent which, whenadmixed with a solution comprising a second solvent, tends to cause thesolute in the second solvent to precipitate. When admixed with acolloid, an anti-solvent may cause flocculation, in analogy withprecipitation of a solute, but admixing a solvent containing dispersednanoparticles and dissolved organic moieties with an anti-solvent maycause, instead of precipitation or flocculation, the self-assembly ofparticles of a different construction, as is described herein. Thelatter are distinguished herein from the “pre-formed” or “pre-existing”nanoparticles that may be incorporated therein. For clarity, the usualimplication that an anti-solvent precipitates a solute, does not obtainherein.

As used herein, the term “surface-modification” refers to a processwherein reactive chemical groups on a surface, in particular the surfaceof a nanoparticle, are added to, removed from, or altered. The modifiedsurface is sometimes referred to as having been “functionalized.”

As used herein, the term “surface-modification” refers to a processwherein reactive chemical groups on a surface, in particular the surfaceof a nanoparticle, are added to, removed from, or altered. The modifiedsurface is sometimes referred to as having been “functionalized.”

As used herein, the term “encapsulation” relates to protectingconstituents of the composite nanoparticle embodiments of the inventionfrom reacting with or diffusing into the medium in which the compositenanoparticle is dispersed. Any such constituent is said to be“incorporated” in the nanoparticle as a “carried agent.” No distinctstructural element is required to confer the protecting function.Similarly, the term “shell” relates herein to the protective function ofa shell and not to any particular structure. The shell of a nanoparticlemay confer hydrophilicity or hydrophobicity on its nanoparticle, may besurface-modified, and may have adsorbed, bound or otherwise anchored toit, without limitation, molecules that interact (bind, react with,complex with) specifically with desired sites in or on materials,including without limitation natural or synthetic fibers, and plant oranimal cells. Such molecules can include, without limitation,antibodies, receptor ligands, and any other means of linking ananoparticle to a site of interest. Nanoparticles so modified are saidto be “targeted” to the desired site, and the antibody, ligand, etc. maybe referred to herein as a “homing” molecule or agent. The shell may beused to affix a “tag” (viz., a means that emits a detectable signal) toa nanoparticle to monitor the whereabouts, the integrity, etc. of thenanoparticle. Alternatively, such a tag may be incorporated into thenanoparticle. As used herein, a “capsule” or “shell” may also have theproperty of allowing encapsulated materials carried in the nanoparticleto be controllably released therefrom.

The term “core,” as it relates to the nanoparticles referred to herein,is a solid-state material which, in its nanoparticle, is not susceptibleto dissolution in the nanoparticle's dispersion medium.

The term “kinematic viscosity” as used herein refers to the tendency ofa fluid to resist flowing (viscosity), factored by the fluid's density.It is the viscosity of a fluid divided by its density.

As used herein, the term “surface plasmon resonance” relates to aphenomenon detectable on metallic surfaces. The “free” electrons thatare characteristic of metals move about within the metal and also on itssurface (as a so-called “plasma” or “electron gas”). Electromagneticsurface waves attend the movement. Because they are “quantized,” thewaves have particle-like properties. The “particles” are called“plasmons.” Plasmons can interact with light, changing its behavior indetectable ways. Under appropriate conditions, the interaction is“resonant.” That is, a plasmon can absorb the light's energy, producingwhat is appreciated by the observer as a “shadow.” These “shadows” are amanifestation of surface plasmon resonance. The phenomenon may be takenadvantage of herein to distinguish metal-containing colloids innanostructures from monomeric metal.

Certain embodiments of the instant invention relate to magneticresonance imaging, especially to agents useful for improving thecontrast between elements of an image. Accordingly, some basicprinciples of magnetic resonance imaging are summarized below to providea better understanding of the utility of these embodiments. Thediscussion is not intended to limit the scope of the invention in any ofits embodiments, especially insofar as any theory may be set forth toexplain how the embodiments are thought to work.

Magnetic materials (iron oxides) are classified by their response to anexternally applied magnetic field, and can be described asferromagnetic, paramagnetic and superparamagnetic (Mornet et al. JournalMaterials Chemistry 2004, 14: 2161-2175; Gupta et al. Biomaterials 2005,26: 3995-4021). A “magnetic material,” broadly conceived, is anymaterial that has—or can be made to have—an electron in motion within,on or around it, inasmuch as a moving electron generates a magneticforce field or “moment.” Since individual atoms typically have at leastone moving electron, they exhibit a magnetic moment or “magneticdipole,” a term that derives from the fact that moments, in addition tohaving magnitude, also have a direction (or orientation). That is, theyare vectors. Ferromagnetic materials are intrinsically magnetic becausethey comprise atoms bound together in a domain, such that the entiredomain becomes a magnetic dipole. Typically, a ferromagnetic materialcomprises many such domains and the extent of their alignment determinesthe magnetic strength of the bulk material. A ferromagnetic materialthat does not appear to be magnetic will become so, permanently, once ithas been exposed, even briefly, to an external magnetic field ofsufficient force and orientation to align all the domains in thematerial. A “paramagnetic” material is different in that it becomesmagnetic only under the influence of an externally applied field (butmay actually increase the force of that field) and rapidly loses itsmagnetization when the external field is removed.

The magnetic properties of a ferromagnetic material can change markedlywhen its bulk is reduced from macroscopic to nanometer scaled particles(Mornet et al. Journal Materials Chemistry 2004, 14: 2161-2175; Gupta etal. Biomaterials 2005, 26: 3995-4021; Pankhurst et al. J. PhysicsD—Applied Physics 2003, 36: R167-R181). Nanometer-sized ferromagneticparticles demonstrate magnetic properties that are characteristic ofneither a collection of “independent” atomic dipoles nor a collection ofdipole domains. In a bulk ferromagnetic material, magnetic domains canbe aligned or “anti-aligned.” The transition between the two domains iscalled a “Block wall.” At the nanometer scale, the formation of Blockwalls becomes thermodynamically unfavorable, leading to the formation ofsingle-domain crystals (Mornet et al. Journal Materials Chemistry 2004,14: 2161-2175; Pankhurst et al. J. Physics D—Applied Physics 2003, 36:R167-R181). These single-domain crystals are no longer ferromagnetic,but exhibit superparamagnetism. For each ferromagnetic material, acritical particle size exists below which domain walls cease to exist.

Superparamagnetic crystals, like paramagnetic materials, losemagnetization when deprived of an external field, but in such a field,they exhibit a much higher magnetic moment. The characteristic strongmagnetic susceptibility (in comparison to paramagnetic materials) ofsuperparamagnetic particles at this scale is a consequence of the singlecrystal nature of the material, which permits the entire crystal toalign with the applied field.

MRI is based on the nuclear magnetic resonance (NMR) signal of protonsin water, lipids, proteins, etc. in tissue, through the combined effectof a strong static magnetic field, B₀, and a transverseradiofrequency-field (also a magnetic field, but oscillatory). Thecounterbalance between the exceedingly small magnetic moment of a singleproton (it is convenient to visualize the magnetic moment associatedwith a proton as a spinning top with a north and south magnetic pole,bearing in mind that the moment of a proton refers to a “field,” not adimensioned structure), and the exceedingly large number of protonspresent in biological tissue, leads to a measurable effect in thepresence of large magnetic fields. For example, for B₀=1 Tesla (T), onlythree of every million proton moments m are effectively aligned parallelto B₀. However, there are so many protons available (6.6×10¹⁹/mm³ ofwater) that the effective signal (2×10¹⁴ proton moments/mm³) isobservable (Pankhurst et al. J. Physics D—Applied Physics 2003, 36:R167-R181). In a clinical setting, the static field, B₀, can be up ashigh as 4 T, while the radiofrequency-field (“RF”) varies between 5-100MHz (Mornet et al. Journal Materials Chemistry 2004, 14: 2161-2175).

The relatively rare aligned proton moments, which spin “straight up” inthe static field except for a slight “wobble” (precession) at acharacteristic frequency (the “Larmor frequency”), are forced by the RFfield to process in resonance with the RF field so that the amplitude ofthe precession increases (the spinning top “leans” severely). Thewobbling magnetic moment is, in effect, an antenna that emits a signal(detectable by MRI machines) when its precession amplitude changes. Whenthe RF field is turned off, the precession of the proton's moment“relaxes” as the static field takes over from the vanishing transversefield. It is important to note that relaxation is the result of twofactors, each of which has its own relaxation time or, more precisely,time constant, referred to as “T₁” and “T_(2.)” The transverse fielddecays rapidly (T₂), whereas the static field reasserts its influenceslowly (T₁) as stored RF energy dissipates into surrounding tissues,which can be visualized as a “lattice” in which the protons areembedded. The reference to “spin-lattice” relaxation derives from thedissipation of the energy stored in the “spin” of the magnetic momentinto the lattice. In contrast, spin energy contributed by the transversefield is “dumped” into the spinning magnetic moment (thus the term“spin-spin” relaxation).

In practice, the time-variant magnetic field (radio frequency transversefield) is applied as a pulsed sequence in a plane perpendicular to B₀and is tuned to the Larmor precession frequency, ω₀, of the proton'smoment in order to get the resonance effect. Despite being much weakerthan B₀, this field has the effect of resonantly exciting the moment'sprecession into the plane perpendicular to B₀, driving a coherentresponse from the net magnetic moment of the protons in the MRI scanner.After the radio frequency sequence is finished, the net magnetizationvector is once again influenced by B₀ and tries to realign with it alongthe longitudinal axis. This relaxation of the coherent response ismeasured via induced currents in pick-up coils in the scanner, which canenhance the signal by a quality factor of approximately 50-100.

In order to correlate the signal to its spatial origin, at least one ofthe two fields (i.e. B₀ or the radio frequency field) has to vary overspace. Relaxation data are collected by a computer which applies atwo-dimensional Fourier transform to give the amplitudes of NMR signalsand permit reconstruction of a 3-D image. Depending on the sequenceparameters, such as the repetition time “TR” (elapsed time betweensuccessive radio frequency excitation pulses) and the delay time “TE”(“echo” time, or the time interval between pulse and measurement of thefirst signal), the desired type of image contrast, T₁ or T₂, can beobtained. In general, short TRs increase T₁ effects, whereas long TRsallow tissues to reach complete longitudinal magnetization, reducing T₁effects. Short TEs minimize T₂ effects of tissues whereas long TEs allowthe loss of transverse signal, enhancing T₂ effects.

Both T₁ and T₂ can be shortened by the use of paramagnetic andsuperparamagnetic contrast agents. This effect is quantified in terms ofthe concentration-independent relaxivities (Bjornerud et al. NMR inBiomedicine 2004, 17: 465-477). The efficiency by which a contrast agentcan accelerate the rate of relaxation of proton moments in a homogeneousmedium is called relaxivity of the agent and is defined by:R _(1,2) =R _(1,2) ⁰ +r _(1,2) C  [1]where R₁=1/T₁ and R₂=1/T₂ are the respective T₁ and T₂ reciprocalrelaxation times (unit s⁻¹) and C is the contrast agent concentration(unit mM). R_(1,2) ⁰ are the relaxation rates in the absence of contrastagent. The slopes of these curves yield the concentration independent T₁and T₂ relaxivities, r₁ and r₂ (unit s⁻¹ mM⁻¹), respectively.

In general, there are two classes of MR contrast agents. On the onehand, there are agents that have low r₂/r₁ ratios, and thereforegenerate positive contrast. For example, the moments of protons inproximity to paramagnetic Gd chelates experience a faster T₁ relaxationthan protons in the absence of such particles. Consequently, differencesin agent concentration result in contrast enhancement on T₁-weightedimages (‘positive’ contrast). On the other hand, superparamagneticnanoparticles produce predominantly T₂ relaxation effects, correspondingto a high r₂/r₁ ratio, which results in signal reduction on T₂-weightedimages (‘negative’ contrast). The phenomenon may be described from thelarge magnetic field heterogeneity around the nanoparticle through whichwater molecules diffuse (Mornet et al. Journal Materials Chemistry 2004,14: 2161-2175). Diffusion induces dephasing of the proton magneticmoments, resulting in T₂ shortening (Mornet et al. Journal MaterialsChemistry 2004, 14: 2161-2175). Increased T₂ relaxivity can be observedat a considerable distance from the nanoparticle, since, in contrast todipolar relaxation, this susceptibility-induced relaxation does notdepend on a direct physical contact between protons and the paramagneticentity. Thus, T₂ shortening can be considered a remote effect, whereasT₁ shortening process requires a close interaction between the watermolecules and the T₁-agents.

Flash nano-precipitation, schematically summarized in FIG. 1, is amicromixing process comprising the steps of dissolving a hydrophobicorganic compound in a compatible solvent, providing a polymer alsodissolved in the solvent or in an aqueous solvent that is ananti-solvent to the organic compound, and rapidly micromixing theorganic solution with the anti-solvent. The materials dissolved in thesolvent(s), upon mixing in the anti-solvent, supersaturate the mixtureand shortly precipitate into a population of unifoitilly sizednanoparticles. The kinetics of the process afford sufficient control toallow the artisan to mix hydrophobic organic compounds with amphiphilicpolymers to produce nanoparticles of predictable size and stability(Johnson et al.,Australian Journal of Chemistry 2003, 56: 1021-1024).The process has been disclosed and described in U.S. Patent ApplicationPublication No. 2004/0091546 and in International Publication No.: WO2006/014626, both of which are incorporated herein in their entirety byreference for all purposes.

In some embodiments, the present invention provides a process comprisingthe steps of dissolving a hydrophobic organic compound in a solvent,dispersing solid inorganic nanoparticles as a colloidal dispersion inthat or another solvent, providing a polymer dissolved in that oranother solvent (which may be an aqueous solvent that is an anti-solventto the organic compound), and micromixing the organic solution, thedispersion and the anti-solvent such that polymeric nanoparticles areformed that retain, sterically stabilized therein, the hydrophobicorganic compounds and the solid inorganic nanoparticles. Organics,including but not limited to organic fluorescent materials andtherapeutic agents such as vitamins, anti-cancer agents, anti-bacterialagents, steroids, or analgesics may be incorporated into the compositenanoparticle. Solid inorganic nanoparticles including but not limited toimaging agents such as iron oxide nanoparticles, gold nanoparticles,gadolinium, and quantum dots may also be incorporated. Because theencapsulating nanoparticles of these embodiments are produced by meansof flash nanoprecipitation, the use of surfactants to stabilize thenanoparticulate dispersions therein is unnecessary, uniformity ofparticle size is intrinsic to the process rather than a consequence ofpost-process purification, and the loading capacity for hydrophobiccomponents is high.

The process of preparing multicomponent composite nano-particles using amulti-inlet or multi-stream vortex mixer is illustrated in FIG. 1. Theco-encapsulation of organic soluble molecules and inorganic colloidalnanostructures is illustrated. Alternatively, a confined impinging jetmixer as described in U.S. Patent Application Publication 2004/0091546(incorporated herein in its entirety by reference for all purposes) canbe employed.

Especially where unequal momentums of the organic and aqueous streamsare advantageous, the multi-stream vortex mixer may be more suitable.Utilization of the multi-stream vortex mixer yields added flexibility insolvent selection, loading of multiple active agents and reduction ofsolvent to anti-solvent ratios. If two (or more) active agents areincompatible together in an otherwise convenient solvent, the two agentscan be mixed from two separate solvent streams, and the velocity of eachstream can be separately controlled. A constant flow rate can beprovided by a syringe pump for each inlet tube using a Harvard Apparatuspump (model number 7023).

An exemplary but non-limiting multi-inlet vortex mixer, made of anyrigid material, comprises a generally cylindrical mixing chamber 0.2333inches in diameter and 0.0571 inches in height. The chamber is definedby a surrounding wall, a first cover or plate sealably disposed inorthogonal relation to the wall and, opposed thereto, a second sealablecover or plate. Four hollow cylindrical inlet tubes, each 0.0443 inchesin diameter, penetrate the wall of the mixing chamber tangentially and,preferably, equidistantly, and are in fluid communication with thechamber. A hollow cylindrical outlet tube, 0.052 inches in diameter, hasits long axis (approximately 0.5 inches in length) disposed inorthogonal relation to the inlet tubes. The outlet tube sealablypenetrates one of the plates centrally and is in fluid communicationwith the chamber.

In some embodiments, a confined impinging jet mixer is suitable. Aconstant flow rate is provided by a syringe pump for each inlet tubeusing a Harvard Apparatus pump (model number 7023). At least one 100 mlglass syringe (SGE Inc.) is connected to each inlet tube. Two solventstreams of fluid are introduced into a mixing vessel through independentinlet tubes having a diameter, d, which can be between about 0.25 mm toabout 6 mm but are between about 0.5 mm to about 1.5 mm in diameter forlaboratory scale production. The solvent streams are impacted upon eachother while being fed at a constant rate from the inlet tube into themixing vessel. The mixing vessel is a cylindrical chamber with ahemispherical top. The diameter of the mixing vessel, D, is typicallybetween 2.0 mm to about 5.0 mm, but preferably is between about 2.4 mmto about 4.8 mm. The mixing vessel also contains an outlet with adiameter, δ, that can be between about 0.5 mm to about 2.5 mm but ispreferably between 1.0 mm to about 2.0 mm. The outlet of the mixer isconnected to an 8-inch line of ⅛^(th)-inch tubing leading out forproduct collection.

The organic solutes, inorganic nanostructures and amphiphilic copolymersare dissolved, solubilized or dispersed, together or separately, in awater-miscible organic solvent including but not limited totetrahydrofuran, dimethyl sulfoxide, or ethanol. Other pharmaceuticallyacceptable water-miscible solvents are listed in U.S. Pat. No.6,017,948, which is incorporated herein in its entirety by reference forall purposes. In preferred embodiments, the inorganic nanostructures(generally sized between 1 nm to 700 nm) are “pre-formed” or“pre-existing” in the sense that they retain their discrete particulatenature when solubilized (dispersed) in the water-miscible organic phaseand continue to retain it after being incorporated into thenanoparticulate product, even if that product simultaneouslyencapsulates organics. Pre-formed nanostructures are not formed duringthe nanoprecipitation process but beforehand.

Intense mixing (i.e., the mixing system operates at a Reynoldsnumber>1600) of the organic solvent stream with water or a predominantlyaqueous stream in the multi-inlet vortex mixer induces, in milliseconds,highly supersaturated mixtures (a solute “supersaturates” a solvent whenthe ratio of the concentration of the solute initially in the mixedstreams in the mixing chamber to the concentration of the solute atequilibrium in the final solvent mixture is greater than 1). The artisancan readily measure the stream velocities of the inlet streams and thekinematic viscosity of each stream by means well known in the art, andcan determine therefrom the Reynolds number for the system, defined asthe sum of the stream flow rates times the average density of the fluidstherein divided by the diameter of the inlet stream and divided by theaverage fluid viscosity of the streams. When solutes mixed under theseconditions precipitate from a supersaturated state, nanoparticles ofuniform size emerge. They are stable and remain dispersed as they leavethe outlet tube. Actives captured within the particles also remainstable.

It is well within the skill of the artisan to “tune” the describedmixing system to cause it to produce nanoparticles of a size between 1nm and 10,000 nm (but preferably less than 1000 nm). A method (“dynamiclight scattering”) for determining sizes of nanoparticles in the contextof the relevant embodiments of the invention is set forth below. Thus,the artisan can select a size distribution that covers a fraction ofthis spectrum by tuning the system through solvent selection, choice ofsolute concentrations, stream velocities, conditions of temperature andpressure, and “time-scaling” as described in detail below.

The stability of the nanoparticles is also within the artisan's control,principally through the selection of polymers. In preferred embodiments,amphiphilic polymers or polymer systems are used. The relative sizes(molecular weights) of their hydrophilic and hydrophobic domainsdetermines stability. The particles tend toward instability ashydrophobic domains are made smaller. As hydrophilic domains are madesmaller, the particles may remain stable internally but, in dispersions,they will tend to aggregate and flocculate.

In polar liquids, charge stabilization (or “electrostaticstabilization”) by Coulombic repulsion is effective. In liquiddispersions, ionic groups can adsorb to the surface of a particle toform a charged layer. To maintain electroneutrality, an equal number ofcounterions with opposite charge will surround the particles and giverise to an overall charge-neutral double layer. The mutual repulsion ofthese double layers provides stability. Charge stabilization, however,is not effective in media of low dielectric constant (the vast majorityof organic solvents and plasticizers) and thus steric stabilization isrequired to maintain the stability of dispersions of the particles.Steric stabilization of the colloid is achievable via attachment ofmacromolecules to the surfaces of the particles in the colloid. Althoughsuch attachment may be covalent in nature, it is typically adsorptive.That is, the macromolecule behaves as if “anchored” on the particle'ssurface but appropriate forces can displace the anchor-point to anothersite on the surface. Steric stabilization would appear to offer severaldistinct advantages over electrostatic stabilization, namely, relativeinsensitivity to the presence of electrolytes in the dispersion media,equal efficacy in both aqueous (polar) and nonaqueous (non-polar)environments, and equal efficacy at both high and low solids content.

In recent years, amphiphilic block copolymers have been demonstrated tobe effective steric stabilizers of colloids. The amphiphilic nature ofthe block copolymer evidently allows one block to have a strong affinityfor the hydrophobic materials in the core of the particle and serves toanchor the copolymer to the particle surface. The second block is morecompatible with the dispersion media and provides a steric barriertowards particle aggregation and flocculation of the colloid.

In some embodiments, the present invention provides a simple process forproducing polymer-encapsulated colloidal particles, each one of whichitself comprises a stable colloidal dispersion. The encapsulatingparticle ranges in size, controllably, from about 25 to about 700 nm.Any colloidal particle dispersion of the present invention will have adistribution of particle sizes for a specific sample. The “size” is thendenoted by one of the moments or averages of that distribution. Thisaverage is calculated by standard dynamic light scattering data analysissoftware such as CONTIN by Brookhaven Instruments, Long Island, N.Y.Alternatively, the size can be determined as the first cumulant of thedistribution as again calculated using commercial dynamic lightscattering software (Brookhaven Instruments, Long Island N.Y.). In thediscussion that follows, if a single size is given it will be the firstcumulant. And if the size distribution is given and an average size isquoted for the distribution it will refer to the light scatteringaverage particle size described below. The process, moreover, affordsthe opportunity to control the degree of stability and thus the specificperformance of products of the process.

In some embodiments, the invention provides a process for preparingcomposite nanoparticles from amphiphilic copolymers. The compositenanoparticles comprise inorganic particles encapsulated in the compositetogether with organic molecules as a colloidal dispersion that iscapable of maintaining sufficient overall stability to accommodate avariety of post-processing manipulations. These manipulations includeaffixing targeting or “homing” molecules to the composite particles, andusing the composite particles to transport molecules and particles totargets. Such molecules or particles may be incorporated in thecomposite particle or affixed, bound, or anchored to the surfacethereof.

Typically, the stabilizing amphiphilic polymer is a copolymer of ahydrophilic block coupled with a hydrophobic block. Nanoparticles formedby the process of this invention can be foimed with graft, block orrandom amphiphilic copolymers. These copolymers can have a molecularweight between 1000 g/mole and 50,000 g/mole, or preferably betweenabout 3000 g/mole to about 25,000 g/mole, and more preferably at least2000 g/mole. Alternatively, the amphiphilic copolymers used in thisinvention exhibit a water surface tension of at least 50 dynes/cm² at aconcentration of 0.1 wt %.

Examples of suitable hydrophobic blocks in an amphiphilic copolymerinclude but are not limited to the following: acrylates including methylacrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate (BA),isobutyl acrylate, 2-ethyl acrylate, and t-butyl acrylate; methacrylatesincluding ethyl methacrylate, n-butyl methacrylate, and isobutylmethacrylate; acrylonitriles; methacrylonitrile; vinyls including vinylacetate, vinylversatate, vinylpropionate, vinylformamide,vinylacetamide, vinylpyridines, and vinyllimidazole; aminoalkylsincluding aminoalkylacrylates, aminoalkylsmethacrylates, andaminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate,cellulose acetate succinate, hydroxypropylmethylcellulose phthalate,poly(D,L lactide), poly (D,L-lactide-co-glycolide), poly(glycolide),poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters),polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethyleneterephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides,polyphosphazenes, poly(amino acids) and their copolymers (see generally,Illum, L., Davids, S. S. (eds.) Polymers in Controlled Drug DeliveryWright, Bristol, 1987; Arshady, J. Controlled Release 17:1-22, 1991;Pitt, Int. J. Phar. 59:173-196, 1990; Holland et al., J. ControlledRelease 4:155-0180, 1986); hydrophobic peptide-based polymers andcopolymers based on poly(L-amino acids) (Lavasanifar, A., it al.,Advanced Drug Delivery Reviews (2002) 54:169-190), poly(ethylene-vinylacetate) (“EVA”) copolymers, silicone rubber, polyethylene,polypropylene, polydienes (polybutadiene, polyisoprene and hydrogenatedforms of these polymers), maleic anyhydride copolymers of vinylmethylether and other vinyl ethers, polyamides (nylon 6,6),polyurethane, poly(ester urethanes), poly(ether urethanes),poly(ester-urea), Particularly preferred polymeric blocks includepoly(ethylenevinyl acetate), poly (D,L-lactic acid) oligomers andpolymers, poly (L-lactic acid) oligomers and polymers, poly (glycolicacid), copolymers of lactic acid and glycolic acid, poly (caprolactone),poly (valerolactone), polyanhydrides, copolymers of poly (caprolactone)or poly (lactic acid) For non-biologically related applicationsparticularly preferred polymeric blocks include polystyrene,polyacrylates, and butadienes. Natural products with sufficienthydrophobicity to act as the hydrophobic portion of the amphiphilicpolymer include: hydrophobic vitamins (for example vitamin E, vitamin K,and A), carotenoids and retinols(for example beta carotene, astaxanthin,trans and cis retinal, retinoic acid, folic acid, dihydrofolate,retinylacetate, retinyl palmintate), cholecalciferol, calcitriol,hydroxycholecalciferol, ergocalciferol, alpha-tocopherol,alpha-tocopherol acetate, alpha-tocopherol nicotinate, and estradiol.The preferred natural product is vitamin E which can be readily obtainedas a vitamin E succinate, which facilitates functionalization to aminesand hyroxyls on the active species.

Examples of suitable hydrophilic blocks in an amphiphilic copolymerinclude but are not limited to the following: carboxylic acids includingacrylic acid, methacrylic acid, itaconic acid, and maleic acid;polyoxyethylenes or polyethylene oxide; polyacrylamides and copolymersthereof with dimethylaminoethylmethacrylate, diallyldimethylammoniumchloride, vinylbenzylthrimethylammonium chloride, acrylic acid,methacrylic acid, 2-acrylamide-O-2- methylpropan sulfonic acid andstyrene sulfonate, polyvinyl pyrrolidone, starches and starchderivatives, dextran and dextran derivatives; polypeptides, such aspolylysines, polyarginines, polyglutamic acids; polyhyaluronic acids,alginic acids, polylactides, polyethyleneimines, polyionenes,polyacrylic acids, and polyiminocarboxylates, gelatin, and unsaturatedethylenic mono- or dicarboxylic acids. The particularly preferredhydrophilic blocks are polyethylene oxide and polyhydroxypropylacrylamide and methacrylamide to prepare neutral blocks since thesematerials are in currently approved medical applications. To prepareanionic copolymers, acrylic acid and methacrylic acid and polyasparticacid polymers are especially preferred, to produce cationic amphiphiliccopolymers DMAEMA (dimethylaminoethylmethacrylate), polyvinyl pyridine(PVP) or dimethylaminoethylacrylamide (DMAMAM).

Preferably the blocks are either diblock or triblock repeats.Preferably, block copolymers for this invention include blocks ofpolystyrene, polyethylene, polybutyl acrylate, polybutyl methacrylate,polylactic acid (PLA), polyglutamic acid (PGA) and PLGA copolymers,polycaprolactone, polyacrylic acid, polyoxyethylene and polyacrylamide.A listing of suitable hydrophilic polymers can be found in Handbook ofWater-Soluble Gums and Resins, R. Davidson, McGraw-Hill (1980).

In graft copolymers, the length of a grafted moiety can vary.Preferably, the grafted segments are alkyl chains of 4 to 18 carbons orequivalent to 2 to 9 ethylene units in length. In addition, the graftingof the polymer backbone can be useful to enhance solvation ornanoparticle stabilization properties. A grafted butyl group on thehydrophobic backbone of a diblock copolymer of a polyethylene andpolyethylene glycol should increase the solubility of the polyethyleneblock. Suitable chemical moieties grafted to the block unit of thecopolymer comprise alkyl chains containing species such as amides,imides, phenyl, carboxy, aldehyde or alcohol groups.

The process yields embodiments of the invention that are productscharacterized by narrow polydispersity and high loading capacity for oneor more active agents stably incorporated in the particles. In oneembodiment, the invention provides a process wherein a population ofhydrophobic pre-existing nanoparticulate constructs, although surroundedby water, do not aggregate into a hydrophobic center, owing to thepresence of an amphiphilic copolymer introduced by the mixing processdescribed herein.

In some embodiments, the pre-existing particles are, surface-modifiedbefore being mixed with polymer and organic moieties. A suitable butnon-limiting method comprises bonding alkyl or aryl phosphonates to thesurfaces of such particles as prescribed in commonly assigned U.S.Provisional Patent Application No. 60/951,113, filed on Jul. 20, 2007. Avariety of surface-modifications are available, any of which may beselected, provided that the modification improves incorporation into thecomposite particle, the stability of the composite particle, the desiredrelease properties or the desired targeting properties of thepre-existing particle (including compatibility at the target-site).

The pre-existing particle is dispersed in a solvent at a controlledtemperature and pressure. An amphiphilic polymer is dissolved in asolvent capable of mixing with the solvent containing the pre-existingparticle, but possessing different solubility characteristics for theamphiphilic polymer. The two solutions are then mixed at a controlledtemperature and mixing velocity, causing selective precipitation of atleast one portion of the amphiphilic polymer or polymer system while atleast one other portion of the same polymer or polymer system remainssoluble. In one embodiment, a product of the process comprises particlesthat have been functionalized by flash precipitation with an amphiphiliccopolymer. In one embodiment, the copolymer is a block copolymer.Preferably, the average size of the functionalized particle is within30% of its pre-process size if single hydrophobic particles are to becoated by the amphiphilic copolymer. The initial size of thefunctionalized particles can be between 50 nm and 50 μm. In a preferredembodiment, the ratio of pre-existing particle to amphiphilic copolymeris 1:1. If the desired composite nanoparticle is to include a pluralityof smaller hydrophobic nanoparticles or hydrophobic nanostructures aloneor in combination with hydrophobic soluble compounds, then the size ofthe resulting composite nanoparticle may be 60% larger to 400 timeslarger than an individual hydrophobic nanoparticle. Most significant tothe stabilization of colloid in the nanoparticles in certain embodimentsof the current invention is the attainment of millisecond micromixing,which is especially advantageous for the steric stabilization ofsub-micron particles.

Pre-existing particles can be comprised of biologically or organicallyactive compounds or precursors including, but not limited toanti-inflammatories, anti-depressants, anti-oxidants, organic andinorganic pigments and dyes, proteins, water insoluble vitamins,fluorescent probes, agricultural actives or precursors, ceramics, latex,glass, or metal. Additionally, in certain embodiments, the presentinvention comprises simultaneously encapsulated hydrophobic and/orelectrostatically charged pre-formed particles along with a dissolvedhydrophobic active agent.

Some illustrative but non-limiting examples are provided herein for thebetter understanding of embodiments of the present invention. In thoseexamples, particle size is characterized by dynamic light scatteringanalysis. The particle sizes are determined from the first cumulant fitof the dynamic light scattering correlation function (West et al.,2003.) The first cumulant fit Γ(q) is expressed as [2],

$\begin{matrix}{{\frac{\Gamma(q)}{q^{2}} = \frac{\sum\limits_{k = 1}^{\max}{n_{k}I_{k}D_{k}}}{\sum\limits_{k = 1}^{\max}{n_{k}I_{k}}}},} & \lbrack 2\rbrack\end{matrix}$where I_(k) is the scattering intensity of particle k, n_(k) is thenumber of particles of a given size, and D_(k) is the diffusioncoefficient of particle k. The scattering wave vector q is given by

$\begin{matrix}{{q = {\frac{4\pi\; n}{\lambda}{\sin( \frac{\theta}{2} )}}},} & \lbrack 3\rbrack\end{matrix}$where n is the refractive index of the solvent, λ the wavelength of theincident light, θ is scattering angle. The first cumulant is related tothe diffusion coefficient by,

$\begin{matrix}{\frac{\Gamma(q)}{q^{2}} = {D_{0}.}} & \lbrack 4\rbrack\end{matrix}$For dilute conditions, the Stokes-Einstein relation applies:

$\begin{matrix}{{D_{0} = \frac{k\; T}{6\pi\;\mu\; a}},} & \lbrack 5\rbrack\end{matrix}$where μ is solvent viscosity. Combining Eqns. 2-5, we obtain anexpression for the scattering intensity weighted radius of theparticles, ā:

$\begin{matrix}{\frac{6{\pi\mu}\;\overset{\_}{a}}{kT} = \frac{\sum\limits_{k = 1}^{\max,\infty}{n_{k}I_{k}}}{\sum\limits_{k = 1}^{\max,\infty}{n_{k}I_{k}\frac{kT}{6\pi\;\mu\; a_{k}}}}} & \lbrack 6\rbrack\end{matrix}$For the particles in the Rayleigh scattering range, where the size ofthe particles is much smaller than the wavelength of scattered light,the intensity of scattered light is proportional to the sixth power ofthe size for each particle.

This leads to the final expression for the diameter obtained by dynamiclight scattering measurements:

$\begin{matrix}{\;{{\overset{\_}{a} \equiv a_{h{\lbrack{6,5}\rbrack}}} = {\frac{\sum\limits_{k = 1}^{\max,\infty}{n_{k}a_{k}^{6}}}{\sum\limits_{k = 1}^{\max,\infty}{n_{k}a_{k}^{5}}}.}}} & \lbrack 7\rbrack\end{matrix}$Therefore, the a_(h[6,5]) moment of the size distribution is theappropriate moment to calculate from the simulations and to compare withthe dynamic light scattering experiments.

Time-scaling. The process depends on tuning three time scales: 1) timeto attain homogeneous mixing (τ_(mix)), 2) time for nucleation andgrowth of the hydrophobic actives (τ_(ng)), and 3) time of blockcopolymer self assembly (τ_(sa)). The process has a characteristicmixing time in the range of milliseconds at a Reynolds number greaterthan 1600.

The mixing time is shorter than the timescale for nucleation and growthof dissolved organic solutes (τ_(ng)). By balancing the nucleation andgrowth times with the block copolymer assembly time, it is possible toblock further particle growth and control nanoparticle size. Too rapidpolymer self assembly consumes the stabilizer and results inuncontrolled growth, while too rapid nucleation and growth results inlargerthan-desired particle sizes. The average nanoparticle size is thuscontrolled by the supersaturation levels and kinetics of aggregation ofboth the block copolymer and hydrophobic compounds.

In one embodiment, the invention provides a composite nanoparticle thatencapsulates polymeric colloidal gold (Au) as an imaging contrast agentand β-carotene as a therapeutic. In one embodiment, the inventionprovides a method of making the composite nanoparticle.

EXPERIMENTAL EXAMPLE 1

Synthesis of Poly(ethylene glycol-block-caprolactone) Block Copolymer

PEG-b-PCL block copolymers were synthesized by acid catalyzedring-opening polymerization of ϵ-caprolactone (PCL) using monomethoxypoly(ethylene glycol) (mPEG) as an initiator according to publishedprocedure (Shibasaki et al., Macromolecules 2000, 33:4316-4320).Dichloromethane and PCL were distilled from calcium hydride underreduced pressure shortly before use. Hydrochloric acid in diethyl etherwas used as received. mPEG (5000 g/mol) was dissolved in tetrahydrofuran(THF), precipitated into cold hexane, and dried under vacuum. Thepolymer was further dried by azeotropic distillation of toluene underreduced pressure. To a solution of mPEG in dichloromethane was addedPCL. Polymerization was catalyzed by addition of hydrochloric acidsolution, and the reaction was carried out at room temperature for 24 h.The copolymer was precipitated into cold hexane, filtered, and dried atroom temperature under reduced pressure. In THF at a concentration of 1mg/ml, the copolymer absorbed no light anywhere in the UV-visiblespectrum.

EXAMPLE 2

Synthesis of Hydrophobic Gold Nanoparticles

Dodecanethiol modified gold nanoparticles (C₁₂—Au) were prepared by atwo-phase reduction of hydrogen tetrachloroaurate (AuCl₄ ⁻) in thepresence of dodecanethiol according to the method of Brust et al.(Journal of the Chemical Society-Chemical Communications 1994,7:801-802. In brief, an aqueous solution of AuCl₄ ⁻ was mixed with asolution of tetraoctylammonium bromide in toluene. The mixture wasvigorously stirred and the organic layer separated. Dodecanethiol wasadded to the organic phase and followed by the addition of aqueoussodium borohydride. The organic phase was separated and evaporated undervacuum. Gold nanoparticles were precipitated into cold ethanol, filteredand dried at room temperature under reduced pressure.

EXAMPLE 3

Preparation of Peg-b-PCL Protected Gold Nanoparticles

A representative synthesis of block copolymer nanoparticlesincorporating pre-formed nanostructures prepared via flashnano-precipitation is as follows. To a solution of PEG-b-PCL(5000-b-6000 g/mole) (55 mg) in THF (HPCL grade) (5 ml) was added dryC₁₂—Au nanoparticles (8.6 mg). The organic solution was fed (12 ml/min,stream 1), along with water (40 ml/min, streams 2-4), into a four-streammulti-inlet vortex mixer (FIG. 1) using two digitally controlled syringepumps (Harvard Apparatus, PHD 2000 programmable, Holliston, Mass.), toyield a final solvent composition of 1:10 v/vol % THF:water. Theconcentrations of C₁₂—Au and PEG-b-PCL in final nanoparticle solutionwere 0.016 wt % and 0.1 wt %, respectively. Nanoparticles were dialyzedagainst Milli-Q water using a Spectra/Por® dialysis bag with MWCO of6,000-8,000 (g/mole) (Spectrum Laboratories Inc., California, USA) andstored at room temperature.

EXAMPLE 4

Characterization

Polymer molecular weights and polydispersity indices were measured bygel permeation chromatography (GPC) using a GPC unit (Waters Inc.,Milford, Mass.) equipped with a series of Phenogel™ columns and adifferential refractive index detector, calibrated with polystyrenestandards (Polysciences Inc., Warrington, Pa.). High-resolution ¹H NMRspectra were obtained using a Varian Inova 400™™ MHz spectrometer.

Nanoparticle size and size distributions were characterized via dynamiclight scattering (Brookhaven Instruments, BI-200SM, Holtsville, N.Y.),consisting of double-pumped continuous NdYAG laser (Coherent Inc.,wavelength 532 nm, 100 mW, Santa Clara, Calif.), and a photomultiplierwith detection angle of 90°. The signal of the photomultiplier wasanalyzed by autocorrelation (ALV-Laser Vertriebsgesellschaft mbH,ALV-5000/E™, Langen, Germany), yielding the time-averaged scatteredaverage particle size and polydispersity index (PDI). The particle sizedistribution was calculated using the ALV-5000/E™ software, from thedecay-time distribution function with the assumption that the scatteringparticles behave as hard spheres (Bohren et al, Absorption andScattering of Light by Small Particles, John Wiley, NY, 1983).

UV-visible absorbance spectra of nanoparticles were collected at roomtemperature using an Evolution 300™ spectrometer (Thermo Electron Inc.,Madison, Wis.) in the wavelength range of 200-800 nm, with a resolutionof 1 nm. Transmission electron microscopy (TEM) images were obtained ona JOEL 2010™ TEM microscope (Tokyo, Japan) working under an accelerationvoltage of 200 kV. For the analysis, a drop of nanoparticles dispersedin water was deposited onto a carbon film supported by a copper grid anddried under reduced pressure. Observations were performed directlyfollowing grid preparation.

Mean particle diameters (hydrodynamic diameters) of PEG-b-PCL-protectedAu particles prepared using the multi-inlet vortex mixer as a functionof Au nano-particle loading are presented in FIG. 2. The error barsrepresent the standard deviation in measured diameters of severalexperimental runs generated at each condition. Nanoparticles wereprepared at fixed block copolymer composition (0.1 wt % in the finalsolution) and Au loading is reported as solids weight percent (Au weightdivided by Au and block copolymer weight). The mean size of unfilledpolymer nanoparticles as prepared in the multi-inlet vortex mixer is50±2 nm. Representative nanoparticles loaded at 7.2 wt % Au are shown ina scanning electron microscopic image in FIG. 11. The term ‘unfilled’refers to nanoparticles prepared using only the block copolymerstabilizer, and which do not encapsulate any Au colloids. The averagenanoparticle diameter is shown to increase with increasing Auconcentration, reaching a value of 103±6 nm at a loading of 23 wt % Au.The inset of FIG. 2 shows the nanoparticle radius, R, normalized by theunfilled micelle radius, R₀, which scales with the gold colloid volumefraction (φ_(Au)) as R/R₀∝(1−φ_(Au))^(−1/3). As elaborated below, theexperimentally observed trend is predicted by a reaction model ofcolloid coagulation in the diffusion limited regime (Fennel-Evans etal., Advances in Interfacial Engineering Series, 2^(nd) ed. 1999,417-424). The corresponding particle size distributions, shown in FIG.3, remain narrow, with PDI values less than 0.25±0.02 obtained in allcases. For reference, particle size distributions of polystyrenecalibration standards of similar sizes (80 nm and 170 nm) are also shownin FIG. 3, with measured PDI values of 0.17±0.03 and 0.13±0.03,respectively.

Since no post-synthesis purification of nanoparticle solutions wasperformed, no material losses are associated with the particlepreparation process and high volumetric productivity is achieved.Typical precipitation processes operate at concentrations below 0.05mg/ml (Kim et al., Langmuir 2007, 23:2198-2202) of the block copolymerstabilizer and often require post processing purification for theremoval of macroscopic aggregates, resulting in significant materiallosses and reduced colloid loadings, on average less than 10 wt % withrespect to the block copolymer (Nasongkla et al., 2006). Using themulti-inlet vortex mixer, nanoparticles with Au loadings of greater than20 wt % have been prepared at block copolymer concentrations in therange of 1.0-4.0 mg/mL, demonstrating both enhanced nanoparticle loadingcapacity and improved volumetric productivity.

A representative TEM micrograph of PEG-b-PCL-protected Au nanoparticlesis shown in FIG. 4. Contrast in the TEM image is provided only by theAu, as the block copolymer is unstained. Individual Au monomers,approximately 5 nm in diameter, are clearly visible. The random, closepacking of the Au monomers within the particle core is evident. For arepresentative Au loading of 23.3 wt %, nearly spherical particles witha mean diameter of 103±6 nm, as determined by dynamic light scattering,are produced. Particle size and size distributions, as determined bydynamic light scattering, are in good agreement with TEM and SEMobservations.

EXAMPLE 5

Simultaneous Loading

The flash nanoprecipitation technology was used to simultaneously loadhydrophobic organic actives and inorganic colloids for integrated drugdelivery and imaging applications. The vitamin A precursor β-carotenewas selected as a model hydrophobic compound and encapsulated, inconjunction with Au, within the cores of PEG-b-PCL nanoparticles usingthe multi-inlet vortex mixer as described. Using ratios ofβ-carotene:Au:block-copolymer of 30.5:5.0:64.5 wt % (fractional weightof β-carotene, Au, and block copolymer with respect to total solidsmass), composite nanoparticles approximately 80 nm in diameter wereprepared. To confirm capture of components within the nanoparticleinteriors, an as-prepared nanoparticle solution was filtered through a10K OMEGA™ nanoseparation centrifuge filter membrane (Pall Corporation,East Hills, N.Y.), which allows for the retention of compositenanoparticles on the membrane surface and permits passage of freeβ-carotene and unprotected Au colloidal particles.

The encapsulation of Au monomers (9.4 wt %) and β-carotene (30.2 wt %)within composite nanoparticles prepared by flash nanoprecipitation wasfirst examined visually as shown in FIG. 5A. The composite nanoparticlesolution prior to filtration was deep red (vial 1), whereas the filtrate(after filtration via a 10,000 MW ultrafiltration membrane) was clear(vial 2). Complete capture of Au and β-carotene is indicated bytransparency of the supernatant (vial 2) when compared to a solution ofnon-protected Au colloid suspended in THF filtered via the same membrane(vial 3). Total recovery of unprotected Au colloid through thenanoseparation filter (vial 3) was confirmed independently viaUV-visible absorbance measurements at 520 nm, where Au colloids of thissize exhibit a maximum in the absorbance spectrum. Transfer ofβ-carotene through the filter was expected based on the molecular weightof the molecule (536.9 g/mol).

Corresponding UV-visible absorption spectra of the compositenanoparticle solution and filtrate are shown in FIG. 5B. A distinctabsorbance peak at 520 nm resulting from the presence of Au colloids isseen in the spectrum of the composite nanoparticle solution, whereasthis peak is essentially undetected in the solution followingfiltration. Quantification of Au concentration in the compositenanoparticle and filtrate solutions was made based on calibratedmeasurements of absorbance values at 520 nm. The UV-visible spectrum ofβ-carotene does not interfere with that of Au in the wavelength range of400-800 nm, and thus the absorbance value at 520 nm can be utilized tocalculate Au concentration in the composite nanoparticle formulation.Based on this calibration, an Au encapsulation efficiency in excess of99.5 weight percent was estimated.

The quantification of β-carotene composite nanoparticle loading iscomplicated by the overlap in absorption spectra of the two componentsin the UV region, where solutions of β-carotene exhibit an absorbancemaximum at 290 nm. As such, we have alternatively prepared PEG-b-PCLnanoparticles in which β-carotene is independently encapsulated.Nanoparticles were isolated as previously described, and theconcentration of free β-carotene in the filtrate measured. Based on UVcalibration at 290 nm, a β-carotene concentration of approximately 0.05mg/mL was estimated. This concentration corresponds to the solubilitylimit of β-carotene in the final solvent composition of 1:10 v/v %THF:H₂O. Thus, all β-carotene in excess of the solubility limit wasincorporated within the nanoparticles. Because nanoparticle loadingrelies on compound solubility, the encapsulation efficiency of organicmolecules will remain unaffected in multiple component formulations.

While the Applicants will not be bound by any particular theory as tothe mechanisms by which any embodiment of the invention works, it isbelieved that the ability of this technology to provide quantitativehomogeneous incorporation of actives arises from the very high level ofsupersaturation of all components, leading to rapid aggregation andcontrolled adsorption of the stabilizing polymer on the compositenanoparticle surface (Brick et al., Langmuir 2003, 19:6367-6380). Thesignificant advantage of the process is that component loadings can beaccurately specified a priori, in contrast to slow, quasi-equilibriumformation processes which lead to unequal incorporation of individualcomponents depending on their solubilities. In our process, drug andimaging agent loadings can be optimized independently and subsequentlyformulated into a single multifunctional delivery vehicle.

EXAMPLE 6

Stability

The extended stability of these particles in the presence ofphysiological salt concentrations was also investigated. Particle sizedistributions, determined by dynamic light scattering, of PEG-b-PCL(5,000-b-6,000 g/mol) nanoparticles encapsulating Au (9.4 wt %) andβ-carotene (30.2 wt %) were stable over time. FIG. 6 shows particle sizedistributions of PEG-b-PCL-protected β-carotene/Au compositenanoparticles immediately after preparation and dialysis and after onemonth of storage in 155 mM saline at room temperature. The mean particlediameter and size distribution of the unfiltered solutions increased toa minor extent from approximately 85 nm to 100 nm over this time,indicating particle stability in aqueous environments for extended timeperiods. The slight increase in particle diameter arises from Ostwaldripening inherent in all nanometer scale particles (Liu et al., PhysicalReview Letters 2007, 98(3))

Nanoparticles in the size range of 100-300 nm are specifically ofinterest, as they have been exploited for passive delivery ofanti-cancer agents to solid tumor vasculature, where defective vasculararchitecture and impaired lymphatic drainage allow for improved particleuptake and localization through the enhanced permeation and retention(EPR) effect (Duncan et al., Annals of Oncology 1998, 9: 39). Wedemonstrate the ability to control the size of composite nanoparticleswithin the above specified range in a predictable fashion in FIG. 7.

PEG-b-PCL-protected Au composite nanoparticles in which the particlesize was ‘tuned’ through addition of an inert component, homopolymer PCL(3,200 g/mol), were prepared. For fixed colloid concentration (0.016 wt% in solution), composite nanoparticle size in the range of 75-275 nm isshown to be a nearly linear function of homopolymer volume fraction(φ_(PCL)) for PCL loadings above 33 vol %. While Applicants will not bebound by any mechanistic explanation of why any embodiment of theinvention works, the relatively constant nanoparticle size with PCLaddition for volume fractions below this value is speculated to resultfrom initial filling of the interstitial voids created by the randompacking of Au monomers in the nanoparticle core, estimated atapproximately 37 vol % (Torquato et al., Physical Review Letters 2000,84:2064-2067). The dense, random nature of monomer packing is supportedby TEM imaging as shown in FIG. 4. PCL addition beyond this pointcontributes to increasing particle diameters. Nanoparticle size andactive loading can thus be specified independently of one another,yielding a highly flexible nanoparticle formation platform.

EXAMPLE 7

Prediction of Encapsulated Colloidal Particle Number and NanoparticleSize

In FIG. 2, the size of polymer-protected Au nanoparticles was shown tobe a function of colloidal particle concentration. In this Example, weillustrate that this behavior is well predicted using a simplerepresentation of colloid self-assembly in the diffusion limited regime,as outlined by Fennell-Evans and Wennerstrom. In this model, a system ofspherical particles each of uniform radius R undergoing Brownian motionis considered. The spheres are assumed to interact according to a squarewell potential of infinite energy with an interaction distance of 2 R.At steady state and in the diffusion limited regime, the rate constantfor colloid association is shown to be independent of aggregate size andcan be used universally to ascertain the kinetics of aggregation,yielding the following general solution to the aggregation process:

$\begin{matrix}{{\lbrack P_{N} \rbrack = {\lbrack P\rbrack_{0}^{tot}( \frac{t}{\tau} )^{N - 1}( {1 + \frac{t}{\tau}} )^{{- N} - 1}}},} & \lbrack 8\rbrack\end{matrix}$where P_(N) is the concentration of particles each composed of Nmonomers, [P]₀ ^(tot) is the monomer concentration at t=0, and τ=2(k[P]₀^(tot)), where k is the universal rate constant given by:

$\begin{matrix}{k \equiv {\frac{8}{3}\frac{k_{B}T}{\mu}}} & \lbrack 9\rbrack\end{matrix}$for which, k_(B) is the Boltzmann constant, T is the solutiontemperature, and μ is the solvent viscosity.

In the multi-inlet vortex mixer, rapid micromixing in the range ofmilliseconds is attained, yielding a homogeneous system in which colloidaggregation and block copolymer precipitation occur in the diffusionlimited regime. In this manner, colloid aggregation persists until blockcopolymer deposition on the assembly surface limits further coagulation.Thus, the time allowed for colloid assembly will be dictated by the sumof the characteristic mixing time in the multi-inlet vortex mixer andthe block copolymer induction time. In the case of PEG-b-PCL, thecopolymer self assembly time is estimated based on a value forcomparable molecular weight poly(ethylene glycol)-b-poly(styrene)(1,000-b-3,000 g/mol) block copolymers as reported in literature, wherethe induction time is approximated to be 37 ms (Johnson et al., 2003).Accounting for an estimated mixing time of 3 ms (Liu et al., ChemicalEngineering Research 2007) in the multi-inlet vortex mixer, snapshots ofthe particle size distributions at a time of 40 ms are calculated usingEq. 8 for varying colloid concentrations (wt % in solution). The resultsof these calculations are shown in FIG. 8. For a given initial monomerconcentration [P₀], the final distribution of aggregates, each composedof N monomers, at an assembly time of 40 ms is calculated. Thenormalized fraction of monomers participating in an aggregate,N*[P_(N)]/[P₀], is shown to reach a maximum for each colloidconcentration studied, with a shift towards larger maximum values as thecolloid concentration is increased. The model additionally predicts acorresponding increase in cluster distribution dispersity as the colloidloading is increased. This trend is supported experimentally, asevidenced by the slightly increasing PDI values of PEG-b-PCL protectedAu nanoparticles with increasing Au content shown in FIG. 3.

Model predictions of cluster sizes calculated at t=40 ms were comparedto particle diameter values, as determined by dynamic light scattering,for PEG-b-PCL protected Au nanoparticles prepared in the multi-inletvortex mixer. For particles in the Rayleigh scattering range, theintensity of scattered light is proportional to the sixth power of thesize for each particle (Bohren et al., 1983). This leads to thefollowing expression for particle radius as obtained by dynamic lightscattering measurements:

$\begin{matrix}{{\overset{\_}{R} \equiv R_{6 - 5}} = \frac{\sum\limits_{N = 1}^{\max,\infty}{n_{N}R_{N}^{6}}}{\sum\limits_{N = 1}^{\max,\infty}{n_{N}R_{N}^{5}}}} & \lbrack 10\rbrack\end{matrix}$where n_(N) is the number of particles of a given radius R_(N). For agiven colloid concentration, particle size can be calculatedanalytically using Eq. 10 in conjunction with the particle sizedistributions, [P_(N)], calculated from Eq. 8. Calculation ofnanoparticle core volume was made assuming each C₁₂—Au monomer occupiesa radius of 4 nm (2.5 nm for Au core and 1.5 nm for C₁₂ extended chainlength). Additionally, packing of the monomers within the nanoparticlecore is assumed to be close packed and random in nature, occupying avolume fraction of 0.63. To account for the PEG-b-PCL stabilizingcopolymer, the diameter of unloaded PEG-b-PCL nanoparticles preparedusing the multi-inlet vortex mixer (50 nm) was added to the clusterdiameters computed through the model.

TABLE 1 Calculated vs. experimental size of block copolymer protected AuNPs prepared via Flash Nanoprecipitation. Calculated average Calculatedaverage number of Au monomers diameter (D₆₋₅) of Experimental diameter(N₆₋₅) in PEG-b-PCL PEG-b-PCL protected of PEG-b-PCL protected protectedAu NPs with σ Au Au NPs with σ in value Au NPs as determined in valuereported as concentration reported uncertainty by DLS uncertainty 0.004wt % 81 ± 3 nm 69 ± 5 nm  38 ± 2 0.008 wt % 89 ± 3 nm 73 ± 6 nm  70 ± 30.016 wt % 98 ± 3 nm 92 ± 5 nm 109 ± 5 0.031 wt % 105 ± 2 nm  103 ± 6nm  126 ± 5

Au concentration in column one is reported as weight fraction of Au insolution. The standard deviations (σ) in calculated and experimentallydetermined values, rounded to the nearest integer, are reported aserror.

Calculated cluster diameters as a function of colloid concentration arereported in Table 1 (column 2). The standard deviation in particle sizecalculated from simulations at each Au concentration is reported as theuncertainties. When compared to experimentally determined particlediameters, as obtained by dynamic light scattering (column 3), theresults show that particle size was well predicted using this simplemodel of colloid aggregation. The model also allows for the predictionof colloid number density within the nanoparticle core. The intensityaveraged particle aggregation number, N₆₋₅ was similarly calculatedaccording to Eq. 10 and the results shown in Table 1 (column 4). Theaverage aggregation number increased with increasing colloid loading,reaching a 126 (σ=5) for the highest Au concentration investigated. Theestimated colloid loading density for this Au composition is wellsupported by TEM imaging for a similarly prepared sample as shown inFIG. 4. This characterization thus allows for accurate a prioridetermination of particle size and colloid loading based solely onprocess inputs, permitting incorporation of multiple inorganic colloidalcomponents at independently specified concentrations.

EXAMPLE 8

Properties of Self Assembled Nanoparticles

Physical properties of colloidal particles are expected to be preservedupon incorporation within the nanoparticle cores. The case of goldcolloid encapsulation shown here is particularly interesting owing tothe electronic behavior of nanometer sized gold crystals. Goldnanoparticles exhibit localized collective oscillation of surfaceconduction electrons, leading to distinctive surface plasmon resonancepeaks in the UV-visible region (Terrill et al., Journal of the AmericanChemical Society 1995, 117:12537-12548). Since the surface plasmonresonance frequency of a particular sample of gold colloid dependsstrongly on the size, shape, dielectric properties, and aggregationstate of the nanoparticles (Link et al., 2000), measuring the phenomenonin encapsulated forms of the colloid is useful in the engineering ofgold-containing nanoparticles.

Encapsulation of Au particles within a block copolymer shell using themulti-inlet vortex mixer was shown to preserve the metallic propertiesof isolated Au nanoparticles. Although Applicants will not be bound byany theory seeking to explain why embodiments of the invention work, itis thought that when Au particles are in close proximity, they are ableto interact electromagnetically, primarily through a dipole-dipolecoupling mechanism. This mechanism broadens and red shifts the plasmonresonance bands (Link et al., 2000). FIG. 9 shows the recordedabsorbance spectra for dispersions of C₁₂—Au in THF and PEG-b-PCLprotected C₁₂—Au nanoparticles in a THF:water:mixture (1:10 v/vol %).The peak in the extinction spectra, lying at approximately 520 nm,remains unaltered in the nanoparticle assembly, suggesting no overlap inthe electronic structure of neighboring Au particles has occurred. Thedodecane capping layer dictates the properties surrounding the goldnanoparticle (medium dielectric constant and refractive index), and itsthickness, estimated between 1-2 nm (Terrill, et al., 1995), controlsthe separation distance between neighboring Au monomers, maintaining theparticles in an electronically independent state. The inter-particleseparation distance, and thus electronic properties of the aggregate,can thus be precisely controlled through selection of an appropriatecapping ligand.

We have utilized this capacity for control over inter-particle distanceto generate composite fluorophore-gold assemblies, in which enhancedfluorescence from the organic dye in the nanoparticle assembly isobserved. This system is expected to provide a photostable imagingplatform with the capacity for particle size control, multi-modalimaging, and reduced toxicity effects.

Finally, we want to place the process of flash nanoprecipitation we haveused here in context with other block copolymer-based nanoparticleformation processes described previously. There are fundamentalthermodynamic constraints which limit the ability of processes used byprevious researchers (Nasongkla et al., 2006; Yang et al., 2007) toproduce multifunctional nanoparticles at high loadings and withcontrolled particle size. Those limitations can be summarized in FIG.10, which shows the precipitation concentrations, or solubilityboundaries, for two components as a function of anti-solvent addition.Previous researchers have slowly added anti-solvent to initially solublesolutions of block copolymers and imaging agents or drugs (Allen et al.,2000; Kim et al., 2001). FIG. 10 displays the solubility of the organicactive β-carotene and the solubility (critical micelle concentration) ofa block copolymer stabilizer (poly(ethylene glycol)-block-polystyrene)as a function of THF content at 35° C. (Johnson, PhD Thesis, PrincetonUniversity, 2003). While the solubility data shown in FIG. 10 isspecific to PEG-b-PS stabilized β-carotene nanoparticles, as detailed inprevious work (Johnson et al., 2003), the operating line shown can begenerally applied to describe the flash nanoprecipitation process. Themethod of slow anti-solvent addition involves traversing the operatingline from the initially pure solvent condition (designated A) in whichall components are soluble, to the intersection with the solubilitycurve for β-carotene (designated B) at 2.5 wt % water in THF. At thispoint β-carotene will begin precipitating. The stabilizing polymer doesnot start aggregating on the particle surface until the waterconcentration reaches 23 wt % (designated C). But at this point over 70%of the β-carotene has precipitated as unprotected crystals. Withoutsubscribing to any theory of why embodiments of the invention work, itis thought, in the case of fast mixing, as achieved by the flashnanoprecipitation process (Johnson et al., AICHE Journal 2003,49:2264-2282), such high levels of supersaturation are produced—inmilliseconds—that, at the final solvent composition (designated D), allspecies aggregate by a diffusion limited, non-specific process. Thecomposition of the resulting nanoparticles reflects the stoichiometry ofthe feeds and no unincorporated material is produced. In this manner,block copolymer nanoparticles at high drug loadings (2.6 wt % PEG-b-PSto 2.6 wt % β-carotene for the case shown) are easily prepared (Johnsonet al., 2003).

Example 9

Nanoparticulate PEG-b-PCL-protected cobalt Ferrite as MRI Contrast Agent

A. Synthesis: All chemicals were purchased from Sigma-Aldrich (St.Louis, Mo.), and unless otherwise noted, used as received. Water,purified by reverse osmosis, ion-exchange, and filtration (Milli-Qwater) was used for particle preparation and dialysis. Oleicacid-stabilized cobalt ferrite (CoFe₂O₄) nanocrystals were synthesizedby Professor Carlos Rinaldi (University of Puerto Rico, Mayaguez).Monomethoxy-terminated poly(ethylene glycol)-block-poly(ϵ-caprolactone)(5,000 g/mole-block-9,000 g/mole; PEG5-b-PCL9) copolymer was synthesizedby ring-opening polymerization of ϵ-caprolactone using mPEG-OH asmacroinitiator and stannous octoanate (SnOct) as catalyst Shuai et al.Macromolecules 2003, 36: 5751-5759.

Block copolymer stabilized CNPs protectively encapsulating cobaltferrite (CoFe₂O₄) nanocrystals were prepared via Flash NanoPrecipitationin a four-stream multi-inlet vortex mixer (MIVM) according to thefollowing protocol: A stock solution of CoFe₂O₄ nanocrytals in hexane(0.3 mL) was dried overnight at room temperature for removal of hexanesolvent. The particles were then re-suspended in tetrahydrofuran (THF; 5mL). To this solution was added PEG5-b-PCL9 (110 mg). The organicsolution was fed (12 mL/min, stream 1), along with water (40 mL/min,streams 2-4), into a 4-stream MIVM using two digitally controlledsyringe pumps. The outlet stream was collected and a sample of theparticle suspension (20 mL) was dialyzed extensively against Milli-Qwater (4 L) for 72 h using a Spectra/Por® dialysis bag with MWCO of6,000-8,000 (g/mole) (Spectrum Laboratories Inc., California, USA) andstored at room temperature. Composite nanoparticle size and sizedistributions were characterized via dynamic light scattering (DLS).Data were analyzed by the cumulant method to determine the hydrodynamicdiameters and particle size polydispersity indices.

Oleic acid-stabilized CoFe₂O₄ nanocrystals of approximately 10 nm indiameter were impingement mixed in the presence of PEG5-b-PCL9 diblockcopolymer. The concentration of block copolymer in all formulations wasfixed, while the concentration of CoFe₂O₄ was allowed to vary,corresponding to the generation of CNPs with increasingly higher loadingof cobalt ferrite. After mixing, particle suspensions were collected,extensively dialyzed against Milli-Q water, and stored at roomtemperature. Samples of the particle suspensions were analyzed forparticle size and particle size distributions via DLS.

Table 2 provides a summary of PEG5-b-PCL9 protected CoFe₂O₄ CNPsprepared in this work. The concentrations of PEG5-b-PCL9 copolymer andCoFe₂O₄ in the final CNP suspension are estimated based on materialconcentrations in the respective inlet streams to the mixer. Theconcentration of PEG5-b-PCL9 is known precisely (column 2). The Feconcentrations (mM) in the final CNP formulations (column 5) areestimated using the concentration of CoFe₂O₄ nanocrystals in the stocksuspension used for CNP preparation, estimated as 23.3 mg/mL based onreagent concentrations used for nanostructure synthesis. Theconcentration of Fe was then based on the 1:2 molar ratio of Co to Fe inCoFe₂O₄ nanocrystals. For reference, the volume of stock suspensionemployed for each CNP formulation is also shown column 3, and can beused to directly calculate the Fe concentration once the ICP assayresults are available. Finally, the mass loading of iron within the CNPconstruct, defined as the mass of Fe to mass of block copolymer, isshown in column 6.

TABLE 2 Compositions of PEG-b-PCL Protected CoFe₂O₄ CNPs Prepared viaFlash NanoPrecipitation ^(a) PEG5-b-PCL9 Stock suspension EstimatedEstimated Fe in final CNP CoFe₂O₄ used for CoFe₂O₄ in CNP content in CNPFe mass suspension CNP preparation suspension ^(b) suspension ^(c)loading Sample (mg/mL) (mL) (mg/mL) (mM) (wt %) ^(d) 1 2 0.10 0.042 0.361.0% 2 2 0.15 0.064 0.54 1.5% 3 2 0.30 0.13 1.1 3.0% 4 2 0.60 0.26 2.26.0% ^(a) Composite nanoparticles (CNPs) prepared in using inlet streamflow rate ratios of 1:10 vol/vol % THF:Milli-Q water, corresponding to atotal CNP suspension volume of 55 mL. ^(b) Concentration of CoFe₂O4 infinal CNP suspension, based on estimated concentration of 23.3 mg/mLCoFe₂O₄ in stock suspension. ^(c) Iron (Fe) concentration based on molarratio of 1:2 cobalt:iron. This value is subject to change pendingresults of ICP assay. ^(d) Ratio of iron mass to mass of blockcopolymer.

The mean intensity-average particle diameters and particlepolydispersity indices (PDI) were determined via dynamic lightscattering (DLS) using the first and second-order cumulants,respectively, of the intensity distributions. CNP diameters and PDIvalues for formulations of Table 2 are reported in Table 3, withcorresponding particle size distributions shown in FIG. 12. The Feloading (wt %), defined as the mass ratio Fe:PEG-b-PCL, for eachformulation is shown in the legend. As can be seen, the meanintensity-average particle diameter is shown to increase with CoFe₂O₄loading from 86 nm for unloaded PEG5-b-PCL9 particles to 360 nm at thehighest CoFe₂O₄ loading investigated. In all cases, particle sizedistributions are unimodal, with PDI values in the range of 0.2-0.25,confirming successful encapsulation of CoFe₂O₄ nanostructures.

TABLE 3 PEG-b-PCL Protected CoFe₂O₄ CNP Size and Particle SizePolydispersity Mean intensity- CNP particle dispersity average CNPdiameter index Sample (nm) (PDI) PEG5-b-PCL9  86 ± 1 0.23 ± 0.02‘unloaded’ particles 1 116 ± 2 0.21 ± 0.02 2 116 ± 1 0.23 ± 0.02 3 175 ±8 0.20 ± 0.02 4  360 ± 10 0.24 ± 0.04

B. ¹H-NMR measurements: The NMR was carried out on a Varian Inova-500NMR spectrometer operating at 500 MHz. For analysis, samples (0.5 mL) ofPEG-b-PCL protected CoFe₂O₄ CNPs (previously dialyzed against Milli-Qfor removal of organic solvent) were filtered via 300 KDa MWCOnanoseparation filters to remove the bulk of liquid (thin film remainson the membrane surface). The retentate was re-suspended in an equalvolume (0.5 mL) of deuterium oxide (D₂O). Using this stock suspension, aseries of samples of increasing dilutions was made using D₂O as diluent.¹H-NMR measurements were performed at 25° C. Typically, T₁ relaxationtimes were determined with an Inversion Recovery sequence on 7 timepoints and T₂ relaxation times were measured with a CPMG sequence withecho time varying from 4 ms to 20 ms on 10 points. All data were fittedwith a mono-exponential curve. The experimental data were fitted by aleast-squares procedure with the expressions:For T ₁ :Y _(i)(t _(i))=A(1−2 exp(t _(i) /T ₁)For T ₂ :Y _(i)(t _(i))=A exp(t _(i) /T ₂)  [11]where t_(i) represent the times at which the magnetization values Y_(i)was measured. The fitting errors were about 1% determined from computerfitting program.

The longitudinal T₁ and transverse T₂ relaxation rates were measured ona ¹H-NMR 500 (500 MHz, 11.75 T, 25° C.). For sample preparation, CNPsuspensions in water were concentrated via 300 kDa MWCO centrifugemembrane to approximately 5% initial volume and re-suspended in equalvolume D₂O prior to ¹H-NMR measurements. Confirmation of CNP integrityfollowing centrifugation and re-suspension cycle was obtained via DLSanalysis.

As shown in FIG. 13 by the DLS spectra of PEG5-b-PCL9-protected CoFe₂O₄CNPs (3 wt % Fe) pre-centrifugation (dashed line) andpost-centrifugation and re-suspension (solid line), the particle sizedistribution of a representative CoFe₂O₄ CNP formulation (3 wt % Feloading) before concentration (dashed line) and following concentrationand re-suspension (solid line) is essentially unchanged, indicatinglittle agglomeration of particles during sample preparation. That is,CNP samples retain their integrity during centrifugation.

FIG. 14 shows the relative sensitivity of translational relaxation rateT₁ and transverse relaxation rate T₂ to the presence of PEG5-b-PCL9protected CoFe₂O₄ CNPs in aqueous media. The Fe loading (wt %) for eachCNP formulation, defined as the mass ratio Fe:PEG5-b-PCL9, is shown inthe legend. FIG. 14a plots the inverse translational relaxation,R₁(1/T₁). FIG. 14b plots the inverse transverse relaxation, R₂(1/T₂).Relaxation rates were measured at 11.75 T and 25° C. Measurements of thereciprocal relaxation rates R₁ and R₂ for CNP formulations at variousdilutions were used to obtain estimates of the concentration independentrelaxivities, r₁ and r₂. The relaxivity values (mM⁻¹ s⁻¹) werecalculated through the least-squares curve fitting of reciprocalrelaxation time versus iron concentration. A summary of results is shownin Table 4.

TABLE 4 Measured r₁ and r₂ relaxivities for PEG-b-PCL protected CoFe₂O₄CNPs Fe(III) Sample loading r₁ R₁ ⁰ r₂ R₂ ⁰ r₂/r₁ 1 1.5 wt % 0.29 ± 0.020.056 ± 0.005 158 ± 7 0.3 ± 2  545 2 3.0 wt % 0.09 ± 0.01 0.065 ± 0.003130 ± 2 3 ± 5 1444 3 6.0 wt % 0.12 ± 0.03 0.067 ± 0.008 195 ± 4 16 ± 121625 Uncertainties in values reported are based solely on linearregression fit of data and do not represent reproducibility inexperimental measurements. Additional experiments to assess experimentalreproducibility are currently under way.

As shown, all CNP formulations have comparable r₁ relaxivity values inthe range of 0.1-0.25 Fe mM⁻¹s⁻¹. These values are significantly smallerthan 20-30 Fe mM⁻¹s⁻¹ typical of hydrophilic commercially availablesingle nanostructure iron oxide particles encapsulated in dextranmatrix, e.g., Clariscan, MION-46 (Ai et al. Advanced Materials 2005, 17:1949—; Wang et al. European Radiology 2001, 11: 2319-2331). The reducedaccessibility of water to CoFe₂O₄ nanocrystals encapsulated within thehydrophobic CNP cores is likely the primary basis for the smaller r₁values obtained in the present case. Because the T₁ shortening processrequires a close interaction between the water molecules and theT₁-agents, it can be inferred that in the present CNP formulations, theinteraction of water molecules with CoFe₂O₄ nanostructures is restrictedto the outer-most layers of CoFe₂O₄ nanostructures accessible within theCNP interior.

By contrast, the r₂ relaxivities are shown to be quite large. For allCoFe₂O₄ CNP formulations examined, the r₂ relaxivity values are withinthe range of 30-100 Fe mM⁻¹s⁻¹, typical of individual, dextran-coatedSPIO particles (Mornet et al. Journal Materials Chemistry 2004, 14:2161-2175; Ai et al. Advanced Materials 2005, 17: 1949—; Wang et al.European Radiology 2001, 11: 2319-2331). Thus, clustering of thesuperparamagnetic nanostructures is shown to result in dramaticincreases in the r₂ relaxivity. The reported values of r₂ relaxivitiesare within the range of other systems which rely on clustering ofsuperparamagnetic nanoparticles to enhance relaxation effects. Forexample, Ai et al. in Advanced Materials 2005, 17: 1949—reportcomparable r₂ relaxivities in the range of 169-471 Fe mM⁻¹s⁻¹ forPEG5-b-PCL5 micelles encapsulating SPIO particles. The authors correlateincreasing r₂ relaxivities with increasing Fe micelle loading. Howeverin their case, the increase in Fe loading was an indirect consequence ofincreasing SPIO particle size (4 nm, 8 nm, and 16 nm) used for micellepreparation. The relaxivity of magnetic nanoparticles has typically beenmodulated by their core size, which are often in the range of 4-20 nm indiameter, with increasing particle size resulting in higher r₂relaxivity values (Wang et al. European Radiology 2001, 11: 2319-2331).

PEG-b-PCL protected CoFe2O4 CNPs (3.0 wt % Fe loading) was synthesizedon three separate occasions to assess reproducibility. The compositionwas strictly maintained for each preparation (constant iron and blockcopolymer loading) and each sample was prepared for NMR analysis. Forall three samples, the T₁ and T₂ relaxation times of each of sixseparate dilutions (iron concentrations) were measured under identicalconditions (11.75 T and 298K). Relaxivity was calculated from relaxationrate and iron concentration data as before. Values for r₁ and r₂ wereobtained from the slope of the least-squares curve fitting of reciprocalrelaxation time versus iron concentration for each sample. They-intercept, R_(1,2) ⁰, represents the inherent relaxation rates of theunloaded PEG-b-PCL particles in the aqueous medium. Values are reportedin Table 5, with corresponding plots shown in FIG. 15.

TABLE 5 ¹H-NMR measured r₁ and r₂ relaxivities of PEG-b-PCL protectedCoFe₂O₄ CNPs of identical composition. Iron Loading Sample (wt %)r₁(mM⁻¹s⁻¹) R₁ ⁰(s⁻¹) r₂(mM⁻¹s⁻¹) R₂ ⁰(s⁻¹) r₂/r₁ 2 3.0 0.090 ± 0.01 0.065 ± 0.003 130 ± 2 3.3 ± 4 1444 2′ 3.0 0.076 ± 0.04 0.067 ± 0.03 153± 3 2.5 ± 4 2013 2″ 3.0 0.084 ± 0.07 0.067 ± 0.05 191 ± 5 3.7 ± 5 2274Reported uncertainty is the errors based solely on the linear regressionfit of relaxation time data and do not represent reproducibility inexperimental measurements.

C. Magnetic resonance imaging: The signal contrast enhancementperformance of the as-synthesized PEG-b-PCL protected CoFe₂O₄ CNPs (3.0wt % Fe, as defined in Table 2) in clinical MR imager (3 T) wasadditionally investigated. PEG-b-PCL protected CoFe₂O₄ CNPs wereprepared for magnetic resonance imaging (MRI) as follows. A 6 wt %solution of gelatin (from bovine skin, Type B) in water was prepared andpoured into an approximately 7″×5″ plastic container. The gel wasallowed to set overnight at 4° C. Sixteen wells, each approximately 1 cmin diameter, were cut out of the mold. A thin gelatin layer (6 wt %) wasthen deposited at the bottom of each well and allowed to set overnightat 4° C. As prepared PEG-b-PCL protected CoFe₂O₄ CNPs in water(following dialysis) were diluted to 60 v %, 40 v %, 20 v %, 10 v %, 6v%, and 2v %. with deionized water. Samples of the CNP suspensions (2 mL)were mixed with gelatin B (120 mg), deposited into individual wells ofthe prepared gelatin mold. Pure solutions of gelatin B (60 mg/mL) in theabsence of CoFe₂O₄ CNPs were also included for reference. The sampleswere allowed to set overnight at 4° C. A final layer of gelatin was thenplaced on the surface and hardened overnight prior to MRI analysis.

FIG. 18a is a photographic image of the prepared gel. PEG-b-PCL CoFe₂O₄samples are placed in wells, arranged from least concentrated to mostconcentrated, with Fe concentrations (mM) as indicated on the figure.For identification purposes, a well containing only dye (dissolved inwater) is added placed ahead of the CNP samples. The reverse mirrorimage of the pattern is repeated to assess reproducibility.

Samples were imaged in a 3T clinical magnetic resonance scanner(MAGNETONM Allegra MR system; Siemens, Malvern, Pa.). For T₂-weightedimaging, a standard spin-echo pulse sequence was used. The followingparameters were adopted: point resolution=0.8×0.8×2 mm, 256×256 bitmatrix, five slices with section thickness=2.0 mm, gap spacing=0.2 mm,TE=15 ms, TR=4000 ms, and number of acquisitions=4. Edge and smoothingfilter (Siemens) was used for image collection.

As seen in FIG. 18b , the PEG-b-PCL protected CoFe₂O₄ CNPs are highlyeffective T₂ MR contrast agents. At the lowest concentrationinvestigated (27 μM Fe), a clear distinction between samples in theabsence of CoFe₂O₄ CNPs and in their presence can be observed. Thus, thepresent CNP constructs can be applied for T₂-weighted MRI contrastenhancement.

EXAMPLE 10

Co-Encapsulation of Organic Active (Drug) and Colloid Contrast Agent

Composite nanoparticles simultaneously encapsulating an organic active(β-carotene) and an inorganic colloidal contrast agent (CoFe₂O₄) wereprepared via Flash NanoPrecipitation. The final composition of the CNPs(by weight percent of total solids) was 32 wt % β-carotene, 64 wt %PEG-b-PCL and 4 wt % CoFe₂O₄, corresponding to a Fe(III) composition of3 wt %. The component compositions of the dually-loaded(CoFe₂O₄/β-carotene) CNP sample along with the singly-loaded (CoFe₂O₄)counterpart are summarized below:

mPEG-b-PCL CoFe₂O₄ β-Carotene Fe (III) concentration concentration,concentration concentration, Fe (III) loading, (mg/mL) estimated(mg/mL)^(b) (mg/mL) estimated (mM)^(c) estimated (wt %)^(d) 2 0.13 0 1.13.0 2 0.13 1 1.1 3.0The intensity-average particle sizes and polydispersity indices (PDI)are reported in Table 6. Uncertainty is reported as the standarddeviation in measured diameters and PDI from five separate DLSexperimental measurements for each sample. The corresponding intensityaverage particle size distributions are shown in FIG. 16. Blue diamondsrepresent CNPs loaded with both CoFe₂O4 and β-carotene, while redsquares represent CNPs containing only CoFe₂O4 colloids. (a) Inverselongitudinal relaxation rates, R₁ (1/T₁) [s⁻¹], and (b) Inversetransverse relaxation rates, R₂ (1/T₂) [s⁻¹], as a function of ironconcentration (mM). The iron loading (wt %) for each CNP formulation,defined as the mass ratio of Fe:PEG-b-PCL, is 3.0 wt % for all threesamples. Trendlines represent the linear regression fit determined byleast-squares curve fitting.

TABLE 6 Particle Size and Polydispersity Indices (PDI) of PEG-b-PCLProtected CoFe₂O₄ and Dually-loaded PEG-b-PCL CoFe₂O₄ + β-carotene CNPsβ-Carotene MNP Hydrodynamic MNP polydispersity concentration (mg/mL)Diameter (nm)^(a) index, PDI^(b) 0 173 ± 2 0.20 ± 0.02 1 173 ± 4 0.24 ±0.01NMR analysis was performed (11.75 T and 298K) to measure T₁ and T₂properties of the dually-loaded composite particles. The relaxationrates (R₁ and R₂) versus iron concentration are displayed in FIG. 17.Blue diamonds represent CNPs loaded with both CoFe₂O4 and β-carotene,while red squares represent CNPs containing only CoFe₂O4 colloids. (a)Inverse longitudinal relaxation rates, R₁ (1/T₁) [s⁻¹], and (b) Inversetransverse relaxation rates, R₂ (1/T₂) [s⁻¹], as a function of ironconcentration (mM). The iron loading (wt %) for each CNP formulation,defined as the mass ratio of Fe:PEG-b-PCL, is 3.0 wt % for all threesamples. The relaxatives, r₁ and r₂, were calculated using aleast-squares curve fitting of these data are summarized in Table 7.

TABLE 7 ¹H NMR measured r₁ and r₂ relaxivities for PEG- b-PCLnanoparticles singly encapsulating CoFe₂O₄ colloid or duallyencapsulating CoFe₂O₄ [B- carotene] (mg/mL) r₁(mM⁻¹s⁻¹) R₁ ⁰(s⁻¹)r₂(mM⁻¹s⁻¹) R₂ ⁰(s⁻¹) r₂/r₁ 0 0.090 ± 0.01 0.065 ± 0.003 130 ± 2 3.3 ± 41444 1 0.089 ± 0.02 0.065 ± 0.002 211 ± 4 1.8 ± 5 2371 The CNP ironloading (wt %), defined as the mass ratio of Fe:PEG-b-PCL is 3 wt % forboth formulations. Reported uncertainty is the errors associated withthe linear regression fit of relaxation time data (FIG. 7) and do notrepresent represent reproducibility in experimental measurements.

The invention claimed is:
 1. A process for manufacturing compositenanoparticles, comprising: a. providing; i) a first water-miscibleorganic solvent comprising a hydrophobic organic compound; ii) a secondwater-miscible organic solvent comprising a colloidal dispersion of aplurality of inorganic hydrophobic nanoparticles selected from the groupconsisting of metallic inorganic hydrophobic nanoparticles and metaloxide inorganic hydrophobic nanoparticles, wherein said inorganichydrophobic nanoparticles comprise a metallic element; iii) a thirdwater-miscible organic solvent comprising an amphiphilic polymer; andiv) an anti-solvent; and b) micromixing said collodial dispersion ofsaid plurality of inorganic hydrophobic nanoparticles in said secondwater-miscible organic solvent with said first and third water-miscibleorganic solvents to create a colloidal composite nanoparticle comprisingsaid metallic element, wherein said colloidal composite nanoparticlecomprises a plurality of inorganic hydrophobic nanoparticle aggregates;wherein said inorganic hydrophobic nanoparticle aggregates comprise asize that is determined by the number of said metallic elements; andwherein said collodial composite nanoparticle comprises a hydrophilicshell and said hydrophobic organic compound.
 2. The process of claim 1wherein said hydrophobic organic compound is insoluble in water.
 3. Theprocess of claim 1, wherein said first, second and third water-misciblesolvents are selected from the group consisting of tetrahydrofuran,dimethyl sulfoxide, and ethanol.
 4. The process of claim 1, wherein saidanti-solvent comprises water.
 5. The process of claim 1, wherein saidamphiphilic polymer is selected from the group consisting of a polymer,a copolymer, block copolymer, graft copolymer, comb-graft copolymer, anda random copolymer.
 6. The process of claim 1, wherein saidinorganic-hydrophobic colloidal particles are surface-modified.
 7. Theprocess of claim 6, wherein said surface-modified inorganic hydrophobiccolloidal particles comprise an electrostatic charge.
 8. The process ofclaim 1, wherein said hydrophobic organic compound comprises anelectrostatic charge.
 9. The process of claim 1, wherein saidmicromixing is performed with a multi-inlet vortex mixer.
 10. Theprocess of claim 1, wherein said micromixing is performed with animpinging jet mixer.
 11. The process of claim 1, wherein said first,second and third water-miscible organic solvents are the same.
 12. Theprocess of claim 1, wherein said first, second and third water-miscibleorganic solvents are different.
 13. The process of claim 1, wherein saidmicromixing comprises flash-nano precipitation.
 14. The process of claim1, wherein said inorganic hydrophobic nanoparticles are selected fromthe group consisting of gold particles, palladium particles, magneticparticles, paramagnetic particles, superparamagnetic metals and oxidesthereof.
 15. The process of claim 1, wherein said inorganic hydrophobicnanoparticles are selected from the group consisting of Gd(III), Mn(II),Mn(III), Cr(II), Cr(III), Cu(II), Fe(III), Pr(III), Nd(III), Sm(III),Tb(III), Yb(III), Ho(III), Eu(II), Eu(III), Er(III), Indium (In),Technetium (Tc), barium particles and quantum dots.